KR102392227B1 - Devices for integrating analyte extraction, concentration and detection - Google Patents

Abstract

Disclosed herein are devices and methods using an aqueous two-phase system and lateral flow assay to detect a target analyte in a sample. Such devices and methods can be used to diagnose a disease or condition in a biological sample, such as blood or serum. In addition, such devices and methods can be used to detect allergens in food samples or contaminants, such as environmental toxins, in water samples. The device and kit components may conveniently be assembled into portable containers and may be suitable for operation in most settings. The device is simple to use, requiring an unskilled operator to simply add a sample to the device. Conveniently, the time taken to detect the target analyte is very short. Accordingly, the devices and methods disclosed herein provide a novel and useful means for management points.

Description

DEVICES FOR INTEGRATING ANALYTE EXTRACTION, CONCENTRATION AND DETECTION

This application claims the following benefits: U.S. Provisional Application No. 61/949,887 (filed on March 7, 2014) and U.S. Provisional Application No. 61/953,870 (filed on March 16, 2014), which is incorporated herein by reference in its entirety.

background

The identification of the unknown, including substances and materials on the one hand and diseases and conditions on the other, is of paramount importance in many different industries. Enzymes and antibodies are used in a variety of contexts including, but not limited to, medical diagnostics, food testing, and environmental contamination ( eg , toxins, pathogens).

summary

Described herein are methods, devices, kits, and systems, including tests performed at a point of care (POC) or point of use, as useful tools for medical, consumer and other uses. In certain applications, points of care or locations of use may include healthcare facilities, mobile clinics, workplaces, factories, farms, and homes. The subject matter described herein is also useful for a variety of other uses, including, but not limited to, air pollutants, toxins, ingredients in food and pharmaceuticals, and industrial, military and space related uses. Among other aspects, a test that is quick, simple, and easy to use is provided herein. The test may, in some cases, be performed by an individual with some clinical diagnostic training. Methods for obtaining test results in a cost and time effective manner are also described herein. Such tests need to be particularly reliable and sensitive to small amounts of biomolecules. In addition, the tests described herein are compatible with many other environments where , for example, temperature and/or humidity vary widely. Accordingly, the point-of-care or point-of-use devices described herein may require a minimal amount of equipment and are stable over a wide range of environmental conditions.

Also disclosed herein are devices and methods comprising an aqueous multiphase system and lateral flow assay (LFA) technology for detecting a target analyte. In certain embodiments, such devices and methods approach or meet, in certain cases, the sensitivity of a laboratory-based assay, such as an enzyme-linked immunosorbent assay (ELISA), at least about 100 to greater than the detection limit of previous LFA assays. It uses the concentration capacity of an aqueous multiphase system with LFA to provide an improvement of about 1000 fold.

Generally, in certain applications, a sample of interest containing the target analyte is subjected to an aqueous two-phase system (ATPS). As described herein, methods and devices have been developed to concentrate and extract a target analyte from a single phase or interface of ATPS with subsequent detection of the target analyte on LFA whose presence can be detected and/or quantified. Alternatively or additionally, the target analyte can be enriched, detected and/or quantified on an integrated ATPS-LFA system. Thus, extraction can be bypassed by seamlessly integrating ATPS with downstream LFA detection. In certain applications described herein, such devices require the user to only add the desired sample. In various embodiments, such devices can be used to detect a target analyte in a biological sample, such as a serum, saliva, urine, blood, or swab sample.

The devices disclosed herein, in certain embodiments, are conveniently assembled to form a portable diagnostic device. The various devices and methods described herein prove to be robust, versatile, scalable, inexpensive, sensitive, simple and accurate, requiring minimal training, power and equipment. It is noted that although the discussion below is generally with respect to aqueous two-phase systems (ATPS), three-phase or four-phase, and even larger phase systems may be similarly practiced.

To illustrate how these concepts function, examples of such techniques are described herein. These examples are intended to be illustrative and non-limiting. Using the teachings provided herein, many other systems that incorporate ATPS and lateral flow detection can be readily implemented. It is understood that the examples and embodiments described herein are for illustrative purposes only, and in light of this, various modifications and changes will occur to those skilled in the art and are to be included within the spirit and scope of the present application and the scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Aspects of the subject matter described herein, including the methods, devices, kits and systems described herein, are devices for detecting and/or quantifying a target analyte in a sample, the device comprising: (a) a lateral flow assay (LFA); and (b) an aqueous two-phase system (ATPS). In certain applications, ATPS comprises a mixed phase solution dividing a first phase solution and a second phase solution. In some embodiments, the splitting of the mixed phase into a first phase solution and a second phase solution occurs within the LFA. In certain embodiments, splitting of the mixed phase into a first phase solution and a second phase solution does not occur in the LFA. In certain applications, the target analyte is contacted with a mixed phase solution, and the target analyte is partitioned into a first phase solution or a second phase solution when/after separation of the first phase solution or the second supernatant. In some embodiments, the target analyte is contacted with the mixed phase solution, and the target analyte is at the interface of the first phase solution and the second phase solution when/after the first phase solution or the second phase solution is separated/separated. is divided In some embodiments, the target analyte is enriched upon cleavage.

In certain embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a polymer. In certain embodiments, the first phase solution comprises a polymer and the second phase solution comprises a micellar solution. In certain applications, the first phase solution comprises a micellar solution and the second phase solution comprises a salt. In certain applications, the first phase solution comprises a salt and the second phase solution comprises a micellar solution. In some embodiments, the micellar solution comprises a nonionic surfactant. In some embodiments, the nonionic surfactant is cetomacrogol, cetostearyl alcorcetyl alcohol, cochamide, decyl glucoside, IGEPAL, isoceteth, lauryl glucoside, monolaurin, nonidet, nonoxynol, NP -40, octyl glucoside, oleyl alcohol, poloxamer, pentaethylene glycol monododecyl ether, polysorbate, polyglycerol, sorbitan, stearyl alcohol, triton-X and tween. In certain applications, the micellar solution is a Triton-X solution. In some embodiments, the Triton-X solution is selected from a Triton-X-100 solution and a Triton-X-114 solution.

In some embodiments, the first phase solution comprises a first polymer and the second phase solution comprises a second polymer. In some embodiments, the first/second polymer is selected from polyethylene glycol and dextran. In some embodiments, the first phase solution comprises a polymer and the second phase solution comprises a salt. In some embodiments, the first phase solution comprises a salt and the second phase solution comprises a polymer. In some embodiments, the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate. In some embodiments, the first phase solution comprises potassium phosphate and the second phase solution comprises polyethylene glycol. In certain embodiments, the first phase solution is selected from component 1 of Table 1 and the second phase solution is selected from component 2 of Table 1 described herein. In certain embodiments, the second phase solution is selected from component 1 of Table 1 and the first phase solution is selected from component 2 of Table 1 described herein.

In some embodiments, the target analyte is selected from a protein, an antigen, a biomolecule, a sugar moiety, a lipid, a nucleic acid, a sterol, and combinations thereof. In certain uses, the target analyte is from an organism selected from the group consisting of plants, animals, viruses, fungi, protozoa and bacteria. In one embodiment, the device further comprises a probe, wherein the probe interacts with the target analyte.

Another aspect of the methods, devices, kits and systems described herein comprises at least one target analyte, or at least two different target analytes, or at least three different target analytes, or at least four different target analytes, or at least 5 different target analytes, or at least 7 different target analytes, or at least 10 different target analytes, or at least 15 different target analytes, or at least 20 different target analytes, or even a greater number of An LFA device comprising one or more probes that interact with different target analytes. In some embodiments, at least 2 different probes, or at least 3 different probes, or at least 4 different probes, or at least 5 different probes, or at least 7 different probes, or at least 10 different probes, or at least 15 different probes Different probes, or at least 20 different probes, are described herein. In certain embodiments, provided are probes described herein comprising a material selected from the group consisting of synthetic polymers, metals, minerals, glass, quartz, ceramics, biological polymers, plastics, and combinations thereof. In certain embodiments, comprising a polymer selected from the group consisting of polyethylene, polypropylene, cellulose, chitin, nylon, polyoxymethylene (DELRIN®), polytetrafluoroethylene (TEFLON®), polyvinyl chloride, and combinations thereof. Probes provided herein are provided. In some embodiments, the polypropylene is polypropylene glycol. In some embodiments, the polyethylene is polyethylene glycol. In certain embodiments, a probe comprising a biopolymer selected from dextran and polyethylene glycol and combinations thereof is provided. In certain embodiments, a probe comprising a metal ( eg , a metal selected from the group consisting of gold, silver, titanium, stainless steel, aluminum, platinum and/or alloys thereof) is provided.

In certain aspects of the methods, devices, kits and systems described herein, probes comprising nanoparticles ( eg , gold nanoparticles) are provided. In certain embodiments, the probe comprises a coating. In various embodiments, the coating is made of polyethylene glycol or other polymers ( eg , polypropylene glycol), or a graft copolymer, such as poly(l-lysine)-graft-dextran (PLL-g-dex, poly(l) -lysine) a graft copolymer having a dextran side chain grafted on the backbone, etc.). In one exemplary, but non-limiting embodiment, the coating comprises dextran. In another embodiment, the coating comprises a hydrophilic protein. In certain applications, the coating comprises serum albumin. In certain embodiments, the coating has an affinity for a first phase solution or a second phase solution. In certain embodiments, a probe comprises a binding moiety that binds to a target analyte. In certain embodiments, the binding moiety is selected from the group consisting of antibodies, lecithins, proteins, glycoproteins, nucleic acids, small molecules, polymers and lipids and/or combinations thereof. In one embodiment, the binding moiety is an antibody or antibody fragment. In certain embodiments, the probe comprises magnetic particles. In certain embodiments, methods, devices, kits, and systems include magnets. In certain embodiments, the magnet is configured to promote and/or increase partitioning of the target analyte into a first phase solution or a second phase solution. In one embodiment, the magnet is configured to facilitate and/or increase flow of a target analyte through the LFA. In certain embodiments, the magnet is attachable to and/or detachable from the device. In certain uses of the methods, devices, kits and systems provided herein, a current collector configured to contact ATPS is provided, wherein a target analyte is partitioned at an interface of the current collector and a first phase solution and/or a second phase solution. do. In some embodiments, the current collector is a plastic, mesoporous material, silica, a polymer ( eg , polypropylene, polyoxymethylene (DELRIN®), polytetrafluoroethylene (TEFLON®), etc.), a magnet, a material having pores, material selected from grooved materials and any combination thereof.

In some embodiments, the methods, devices, kits and systems described herein comprise a probe comprising a detectable label. In some embodiments, the detectable label is selected from the group consisting of a colorimetric label, a fluorescent label, an enzymatic label, a colorigenic label, a radioactive label, and combinations thereof. In some embodiments, the LFA comprises a porous matrix. In certain applications, the porous matrix is sufficiently porous to allow the mixed phase solution, the first phase solution, the second phase solution, and/or the target analyte to flow through the LFA. In certain embodiments, long enough and/or deep enough to allow the mixed phase solution, the first phase solution, the second phase solution, and/or the target analyte to flow vertically and/or horizontally through the LFA, and any combination thereof. A porous matrix provided herein is provided. In some embodiments, a method, device, kit or system is provided wherein a first phase solution flows through the porous matrix at a first rate and a second phase solution flows through the porous matrix at a second rate, wherein the first The speed and the second speed are different. In certain embodiments, the porous matrix comprises a material selected from cellulose, glass fibers, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and combinations thereof. In some embodiments, an LFA comprising a target analyte capture moiety is provided, wherein the target analyte capture moiety interacts with a target analyte. In certain embodiments, LFAs comprising a competition assay are provided. In certain embodiments, an LFA comprising a target analyte is provided. In certain embodiments, methods, devices, kits and/or systems using LFA comprising a sandwich assay are provided. In certain embodiments, the LFA comprises a probe capture moiety, wherein the probe capture moiety interacts with the probe or component thereof. In certain embodiments, the mixed solution phase solution is dehydrated on and/or within the LFA strip, and upon addition of the sample, the mixed phase solution is partitioned into a first phase solution and a second phase solution.

In certain embodiments of the methods, devices, kits and/or systems described herein, LFAs comprising wells having a volume sufficient to contain a solution are provided. In certain applications, the solution is selected from the group consisting of: at least a portion of ATPS, at least a portion of a first phase solution, at least a portion of a second phase solution, a resuspended solution of a target analyte, and combinations thereof. In further or additional embodiments, an LFA comprising a well is provided, wherein the well is disposed at a location of the LFA selected from corner, distal, central, junction, off-center and bend. In some embodiments, a well comprises one or more pads selected from a filter pad, a buffer pad, a surfactant pad, a salt pad, a probe pad, a polymer pad, and combinations thereof. In certain embodiments, an LFA comprising a strip is provided. In a further or additional embodiment, provided that the strip is constructed according to a structure selected from the structures shown in FIGS. 16 , 20 and 62-70 . In further or additional embodiments, an LFA is provided wherein the strip comprises a multi-path route. In certain applications, the LFA further comprises a dry destination. In some embodiments, the device comprises a working buffer. In some embodiments, a device comprising a port for administering a sample ( eg , a biological sample) to the device is provided. In certain applications, ports connected to ATPS are provided. In some embodiments, ATPS and LFA are integrated, and when a port is connected to an LFA, an LFA is provided. In certain embodiments, the port comprises a structure selected from a tube, a funnel, a valve, a syringe, a straw, a channel, a plunger, a piston, a gravity feeder, a pump, and combinations thereof. In some implementations, the device does not require a power supply. In certain embodiments, ATPS and LFA are integrated prior to use of the device. In further or additional embodiments, ATPS and LFA are separated prior to use of the device. In some embodiments, the device is configured to insert LFA into ATPS.

In certain aspects, the methods, devices, systems and/or kits described herein comprise or use a device comprising: (a) a first component comprising a chamber for containing ATPS; and (b) a second component comprising LFA. In one embodiment of this aspect, a device comprising an actuator that delivers a sample and/or a target analyte, or both, to ATPS is provided. In certain embodiments, a device is provided that further comprises an actuator that delivers a solution to the LFA. In certain applications, an LFA comprising a solution selected from the group consisting of a mixed phase solution, a first phase solution and a second phase solution, and combinations thereof is provided. In certain embodiments, ATPS and LFA are contained in a single housing. In certain applications, a portable LFA device is provided.

In certain aspects, methods, devices, systems and/or kits are provided for detecting and/or quantifying a target analyte in a sample, wherein the detection and/or quantification comprises: (a) according to as described herein applying a sample to the device; and (b) detecting the presence or absence of the target analyte on the LFA. In certain embodiments, the method comprises subjecting the sample to ATPS. In certain applications, the method comprises subjecting a sample to LFA, wherein the LFA and ATPS are integrated. In certain embodiments, the method comprises enriching the target analyte in ATPS. In one embodiment, the method comprises enriching the target analyte in LFA. In some embodiments, the methods include tissue/fluids, food samples, chemical samples, drug samples, and environmental samples ( eg , water samples, soil samples, etc. ) from biological organisms ( eg , plants, animals, algae, fungi, etc. ); and one or more samples selected from the group consisting of combinations thereof. In some embodiments, a sample selected from the group consisting of a blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample, a bronchoalveolar lavage fluid, a nasal sample, a fecal sample, a sample from a wound or surgical site, and combinations thereof is provided do. In certain embodiments, the target analyte comprises a biological molecule. In certain embodiments, the biological molecule is selected from the group consisting of nucleic acids, proteins, lipids, small molecules, sugars, antibodies, antigens, enzymes, and combinations thereof. In certain embodiments, the sample is from a vertebrate ( eg , mammal, avian , etc. ), bacteria, virus, protozoan, avian, fungus, protozoan, drug, pathogen, toxin, environmental contaminant and components thereof, and their components. from a source selected from the group consisting of combinations.

In certain aspects, methods, devices, kits and/or systems using a paper fluidic device for detecting a target analyte in a sample are provided, wherein the paper fluidic device is used in conjunction with an aqueous two-phase system (ATPS). In certain embodiments, a device comprises a porous matrix disposed with ATPS or a component thereof, wherein the porous matrix is configured such that the ATPS or component thereof flows through the porous matrix when the ATPS or component thereof is in a fluid phase, has sufficient porosity.

In certain embodiments, the paper fluid device comprises ATPS or a component thereof. In certain embodiments, ATPS or a component thereof is selected from the group consisting of a first phase solution, a second phase solution and a mixed phase solution, wherein the mixed phase solution is a first phase solution and a second phase solution and/or a combination thereof. contains a mixture of In certain embodiments, ATPS and/or components thereof are dehydrated on and/or within at least a first portion of the porous matrix. In certain applications, paper fluid devices, methods, systems, and/or kits are provided wherein a first portion of the porous matrix has a different width than a second portion of the porous matrix. In further or additional embodiments, a paper fluidic device, method, system and/or kit is provided for providing ATPS or a component thereof in a fluid phase using a sample to dehydrate ATPS. In some embodiments, the paper fluid device comprises a well for containing a content selected from the group consisting of a mixed phase solution, a first phase solution, a second phase solution, a sample, and combinations thereof. In certain applications, the paper fluid device includes an actuator for releasing the contents of the well into and/or onto the porous matrix. In certain embodiments, a well comprises one or more pads selected from a filter pad, a buffer pad, a surfactant pad, a salt pad, a probe pad, a polymer pad, and combinations thereof. In some embodiments, the first phase solution and the second phase solution flow through the porous matrix at different rates. In one embodiment, the first phase solution and the second phase solution flow through the porous matrix in different directions. In certain embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a polymer. In a more specific embodiment, the first phase solution comprises a micellar solution and the second phase solution comprises a salt. In certain applications, a paper fluid device comprising a Triton-X (or other surfactant) micellar solution is provided. In some embodiments, the first phase solution comprises a polymer and the second phase solution comprises a polymer. In certain embodiments, the first phase solution comprises the polymer and the second phase solution comprises the salt. In certain embodiments, a paper fluid composition is provided wherein the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate.

In one aspect, there is provided a method, apparatus, system and/or kit comprising a paper fluid device wherein the first phase solution is selected from component 1 of Table 1 and the second phase solution is selected from component 2 of Table 1 . For certain applications, devices, methods, systems and/or kits are provided that are constructed according to the structures shown in FIGS. 16 , 20 and 62-70 . In certain applications, a porous matrix comprising a first pathway and a second pathway is provided.

In another aspect, methods, devices, systems, and/or kits comprising or using a paper fluidic device are provided, wherein a first phase solution preferentially flows through a first pathway and a second phase solution preferentially flows through a first pathway. 2 flows through the path. In some embodiments, a paper fluidic device is provided wherein the composition/device comprises a probe that binds to a target analyte to produce a probe-analyte complex. In certain embodiments, provided is a paper fluid composition wherein, in use, a target analyte binds to a probe in a probe-analyte complex. In some embodiments, a probe comprising magnetic particles is provided. In certain embodiments, the device comprises a magnetic field oriented to attract magnetic particles to a portion of the porous matrix and/or the method uses such a magnetic field, wherein the force of the magnetic field on the magnetic field particle is probe-analyzed toward a portion of the porous matrix. Improves the flow of the water complex. In certain embodiments, comprising a polymer selected from the group consisting of polyethylene, polypropylene, nylon, polyoxymethylene (DELRIN®), polytetrafluoroethylene (TEFLON®), dextran and polyvinyl chloride, and combinations thereof. A probe is provided. In some embodiments, the polyethylene is polyethylene glycol. In some embodiments, the polypropylene is polypropylene glycol. In one embodiment, a probe comprising a biological polymer selected from the group consisting of collagen, cellulose and chitin is provided. In certain applications, a paper fluidic device is provided wherein the probe comprises a metal selected from the group consisting of gold, silver, titanium, stainless steel, aluminum, platinum, alloys thereof, and/or combinations thereof. In one embodiment, a probe comprising nanoparticles ( eg , gold nanoparticles) is provided. In certain applications, the probe includes a coating. In certain embodiments, the coating comprises polyethylene glycol. In one embodiment, the coating comprises dextran. In one embodiment, the coating comprises polypropylene. In one embodiment, the coating comprises polypropylene glycol. In certain embodiments, the coating comprises a hydrophilic protein. In certain embodiments, the coating comprises serum albumin. In certain applications, paper fluid compositions/devices are provided wherein the probe has a coating having an affinity for either a first phase solution or a second phase solution.

In certain aspects of the methods, devices, systems and/or kits described herein, a paper fluid composition/device comprising a probe is provided, wherein the probe further comprises a binding moiety that binds to a target analyte. In some embodiments, a paper fluidic device is provided comprising a binding moiety selected from the group consisting of antibodies, lectins, proteins, glycoproteins, nucleic acids, small molecules, lipids, and/or combinations thereof. In certain embodiments, a probe comprising a binding moiety comprising an antibody or antibody fragment is provided. In certain embodiments, a paper fluid device comprising a probe comprising a detectable label is provided. In one embodiment, the detectable label is selected from the group consisting of a colorimetric label, a fluorescent label, an enzymatic label, a color genic label, a radioactive label, and an assembly thereof. In certain applications, a paper fluid composition further comprising a dry destination is provided, wherein the first phase solution or the second phase solution preferentially flows through the porous matrix towards the dry destination.

In certain embodiments, the methods, devices, systems and/or kits described herein comprise a paper fluidic device/composition comprising, in use, a working buffer, wherein the first phase solution and/or the second phase solution is contacted with the working buffer. flow more rapidly through the porous matrix. In certain uses of the subject matter described herein, a porous matrix comprising a material selected from cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and/or combinations thereof A paper fluid device is provided. In one embodiment, a paper fluidic device comprising a porous matrix comprising a target analyte capture moiety is provided, wherein the target analyte capture moiety interacts with a target analyte or component thereof. In one embodiment, the paper fluidic device further comprises a target analyte. In one embodiment, the porous matrix comprises a probe capture moiety, wherein the probe capture moiety interacts with the probe or component thereof. In certain embodiments, LFAs comprising or facilitating competition assays are provided. In certain embodiments, the LFA comprises a sandwich assay. In certain embodiments, a paper fluid composition is provided, wherein the porous matrix is configured to concentrate the target analyte as it flows through the porous matrix. In certain applications, the paper fluidic device comprises a control analyte, wherein comparison of the control analyte and the target analyte on the porous matrix provides for quantification of the target analyte.

In certain aspects, a method of detecting a target analyte in a sample is provided, the method comprising: (a) applying the sample to a device described and/or claimed herein; and (b) detecting the presence or absence of the target analyte.

Brief description of the drawing

The above summary as well as the following detailed description may be better understood when read in conjunction with the accompanying drawings. The drawings are intended to be illustrative and not restrictive. It will be understood that the present disclosure is not limited to the precise arrangements, examples and instrumentalities presented herein.

1 (Top) Schematic of a typical lateral-flow immunoassay test strip. Two exemplary mechanisms used by LFA are presented: a sandwich assay (lower left) and a competition assay (lower right).

2 is a schematic for analyte concentrations using ATPS and capture probes. is also representative. The probe captures the target analyte and splits it into one of the extreme phases. The phase containing the enriched probe can then be collected for subsequent analysis via LFA.

3 shows a schematic representation for analyte concentrations using ATPS and magnetic capture probes. The probe captures the target analyte and splits it into one of the phases. A magnet is applied which further concentrates the magnetic probe to a smaller volume.

4 depicts one exemplary, but non-limiting design of a portable device that uses a piston and plunger to move concentrated analyte from ATPS to an LFA test strip. This device consists of a concentration chamber and a detection chamber. In this example, the analyte is concentrated on the lower bulk of ATPS. To collect the sample, (1) compress the plunger. The sample is mixed with the ATPS solution. Subsequently (2), after inserting the tip of the device into the sample to withdraw the solution, (3) releasing the plunger, and (4) sealing the PEN device with a cap. (5) After concentration of the analyte in the lower bulk phase, the detection step includes (6) rotating the plunger in the “TEST” position (which lowers the piston), and (7) directing the concentrated analyte through the check valve to the detection chamber. It starts by inducing (8) Adjust the capture lock in the groove to ensure the movement of the piston, and only the adjusted amount of volume is introduced into the detection chamber.

5 depicts one exemplary, non-limiting design of a portable device for mechanically inserting an LFA test strip onto a top containing a concentrated analyte. To draw the sample premixed with the ATPS solution, press the (1) button on the side of the device. After completing phase separation and concentrating the analyte on the upper bulk, the detection step is initiated by pressing the (2) detection button which lowers the LFA test strip so that the strip's sample pad is in contact with the concentrated analyte. Finally, (3) results can be interpreted through the results window.

6A-D show an exemplary, but non-limiting design of a portable device for enforcing a sealed well (A). ATPS solution is mixed in the well. After phase separation is complete, the flow of the well can be captured by puncturing a seal at the bottom of the well, and the lower phase containing the concentrated analyte flows towards detection. This approach can be incorporated into devices that concentrate the analyte on top or bottom. Once the analyte is concentrated in the lower phase, the well is recessed in the device chamber to capture the flow of the lower phase towards detection. Alternatively, the wells can be loaded from below and a button added to the well is introduced onto the device. When this button is pressed, it may cause the test strip to contact the well. 6B and 6C show the following results: 1:1 volume PEG: salt ATPS and 9:1 volume PEG: salt ATPS on test strips. 6D illustrates an exemplary mechanism for puncturing the well and capturing the flow of ATPS solution(s) through the LFA: retract the button in contact with the well (lower panel) or retract the well itself (upper panel) .

7 depicts one exemplary, but non-limiting design of a portable device. This apparatus is similar to the apparatus presented in Fig. 4 , but uses a magnetic probe. During the formation of the lower phase, a magnet near the cap of the pen further concentrates the magnetic probe into a smaller volume. When the button on the pen is retracted, the magnetic particles are flushed through the check valve to initiate detection.

8 shows a schematic representation of the thickening phenomenon at the interface. Due to the specific properties of the probe and ATPS, the probe is cleaved at the interface and is highly concentrated. The interface can then be collected for subsequent analysis via LFA.

9 shows the observation of solid-liquid interface concentration. The probe was found to be present on the polypropylene wall of the tube containing the PEG-salt ATPS.

Figures 10a-b : (a) depict experimental collection of analyte-probe complexes using polypropylene sticks in PEG-salt ATPS. (b) A schematic representation of the solid-liquid interface concentration is shown. The probe binds to the target analyte in a homogeneous solution. Since the wall of the tube is glass, the probe is preferentially attached to a polypropylene collector inserted into the TPS. The collector containing the enriched probe can be removed and the probe can be resuspended for detection via subsequent LFA. Note that these particles are attached to the solid propylene only when a two-phase system is present.

11 shows a schematic representation of a portable device using a solid-liquid interface concentration. ATPS samples are added to the inserted collection stick to collect the concentration wells and probes. After the probe is attached to the collection stick, it is transferred to a detection well that resuspends the probe and captures the flow towards the LFA for detection.

12a-c show (a) that the paper causes ATPS phase separation to occur when it flows. Polymer-salt ATPS is used in this example. The bulk components of the hydrophobic and coercive viscous phases are delayed later, while the hydrophilic and less viscous phases containing the concentrated target analyte flow rapidly through the paper. (b) 1:1 polymer-salt ATPS phase separates from the paper and flows through the paper within 5 minutes. (c) 9:1 PEG-salt ATPS also flows through the paper within 5 minutes, but the capture probe in the polymer-deficient phase is not readily visible due to its smaller volume.

13a-c show (A) paper wells enable more efficient ATPS phase separation. The bulk components of the hydrophobic and more viscous phases are retained by the paper and remain in the upper layers of the wells, while the hydrophilic and less viscous phases containing the target analytes flow rapidly to the lower layers. To enhance the collection of analytes, addition of working buffer can be applied to flush from paper wells on the membrane while maintaining phase integrity. 13B shows that the 1:1 PEG-salt ATPS phase separates in a layer of paper and flows through the membrane for detection. 13C shows that 9:1 PEG-salt ATPS flows through the membrane more slowly due to the PEG rich phase containing large PEG molecules at high concentrations. The concentrated salt phase then becomes the leading edge.

14A-D show (A) variation of 3D paper wells, where we use both concentration wells and mixed wells. To start the assay, samples mixed with ATPS solution are applied to concentration wells. (B) Buffer is then added to the concentration wells to facilitate phase separation. (C) Once the concentrated analyte reaches the mixing well, (D) another buffer is added to the mixing well to facilitate flow and allow the concentrated analyte to reach the LFA test strip downstream.

15a-b demonstrate a method for dehydrating the components required for ATPS in a paper strip capable of inducing phase separation after the components are rehydrated into a sample solution (a). In this experiment, the sample solution consists of a blue dye, favoring a polymer rich phase, and a red colorimetric nanoparticle indicator for LFA, favoring a polymer-poor phase. FG stands for fiberglass paper. (b) Control condition without the dehydrated ATPS component.

16A-E illustrate different paper device structures using a dehydrated PEG-salt ATPS component. (a) Simple structure with separate PEG and salt segments. (b) Simple structure with combined PEG and salt segments. (c) Example of introducing a plurality of repeating segments to improve enrichment efficiency. (d) Repeat of example c wherein the PEG segment is not present in the direct flow path of the device. (e) An example of a structure arranging a phase separation site at the end of a flow path. An LFA test strip, not shown in this schematic, is placed in the downstream area of the device.

17A- B show (A) PEG-salt paper- which can be used to further concentrate the fluid by drawing a solution from the dehydrated PEG segment to form a PEG-rich phase excluding the desired analyte based on size exclusion; A schematic diagram illustrating a method of offsetting a dehydrated PEG segment within a unique diagnosis; and (B) experiments demonstrating the enrichment effect.

18A-B are (A) schematic representations of introducing a working buffer to drive the retardation phase containing concentrated analyte towards LFA test strips; and (B) an exemplary alternative design for introducing a working buffer to drive the reading phase containing the concentrated analyte towards the LFA test strip.

19 shows a schematic representation of a flow rerouting design using 3D paper wells with different paper materials. The polymer-rich phase is more hydrophobic and preferentially flows through the more hydrophobic paper, while the polymer-depleted phase flows more preferentially through the hydrophilic paper. An LFA test strip can be attached to the region downstream of this device for detection.

20 illustrates a different example of a paper device layout that introduces the use of magnetic probes to enhance enrichment capacity. Here, the position of the magnet provides a force acting on the particle bound to the target analyte. This effectively concentrates the particles on the side of the paper with the LFA detection area.

Figures 21a-c show that GMP was found to be extremely cleaved on the underlying PEG defect (a). This enables the use of a small magnet (b) to quickly recover most of the GMP from the ATPS solution (c).

22 shows images of LFA strips used to detect Tf with (top) or with (bottom) no prior ATPS enrichment step.

23A-D depict images of PGNP and PP straws in solution containing (a) PEG alone, (b) salt alone or (c) 1:1 ATPS. PGNP was highly cleaved in PP straws only in the presence of ATPS. (d) PP straws were extracted from (c), which shows that the straws can be withdrawn to facilitate collection of concentrated PGNP for subsequent detection assays.

24 shows images of LFA strips used to detect transferrin (Tf) without (top) and with (bottom) a prior ATPS enrichment step.

25 shows a schematic representation of the proposed portable device.

26 depicts a schematic diagram of DGNPs and their role in an approach to improve the detection limit of LFA using ATPS.

27 depicts a schematic representation of positive and negative results for a competition test of LFA.

28A-C show normalized visibility of (a) citrate capped or stripped gold nanoparticles (b) PEGylated gold nanoparticles, and (c) dextran coated gold nanoparticles in varying % w/w potassium phosphate solutions. The ray range absorbance spectrum (400-700 nm) is shown.

29A-C depict the cleavage behavior of DGNP in PEG-salt ATPS viewed at (a) 0 min, (b) 30 min and (c) 12 h.

30 depicts experimentally determined Tf concentration factors at various time points within 12 hours in PEG/salt ATPS. Error bars represent standard deviation from triplicate measurements.

31A-B show images of LFA strips used to detect Tf without (a) and with (b) a pre-concentration step using PEG/salt ATPS.

32 depicts the results of LFA quantification using MATLAB. Error bars represent standard deviation from measurements using three LFA test strips.

33A-D depict brilliant blue FCF dye and dextran coated gold nanoparticles that are highly partitioned into upper PEG-rich and lower PEG-deficient phases, respectively. (a) At 25° C., the mixed 1:1 volume ratio ATPS phase separates to form (b) two equal volume phases. (c) At 25 °C, the mixed 9:1 volume ratio ATPS phase separates into (d) a large PEG rich phase and a small PEG deficient phase. Equal amounts of Brilliant Blue FCF and dextran coated gold nanoparticles were added to both ATPSs. The dark purple color of ATPS in a 9:1 volume ratio indicates a significant increase in gold nanoparticles concentration in the underlying PEG-deficient phase.

34a- b show that the paper membrane allows ATPS phase separation to occur as it flows. The PEG-rich domains remained near the tip of the paper membrane, while the PEG-deficient domains moved rapidly to the front of the reading. (a) 1:1 volume ratio ATPS phase separated and flowed through the paper membrane within 5 min. (b) 9:1 volume ratio ATPS also separated and flowed through the membrane within 5 min, but the phase separation was less efficient because the leading front was less distinct at 300 s.

35A-B show that 3-D paper wells enable further enhanced ATPS phase separation. After addition of the dye and mixed ATPS, the PEG-rich domains remained in the upper layer of the well, while the PEG-deficient domains containing the enriched gold nanoparticles flowed rapidly into the lower layer. (A) 1:1 volume ratio ATPS showed enhanced phase separation in 3-D paper wells, and the PEG-deficient phase flowed through the membrane within 5 min. (b) 9:1 volume ratio ATPS also had improved phase separation, and the PEG-deficient phase was clearly visible. In contrast to: FIG. 34 , the leading front surface is well defined and has a deep purple color indicating the concentration of dextran coated gold nanoparticles.

36A-C show 3-D paper wells were combined with PEG/salt ATPS and LFA for Tf detection. (A) Paper well devices were combined with a Tf competition assay on nitrocellulose paper. Samples containing no Tf were diagnosed correctly when using (b) 1:1 or (c) 9:1 volume ratio ATPS solutions with visual test and control lines.

37A-B show that a 1:1 volume ratio PEG/salt ATPS with 3-D paper wells allows for a 2-fold improvement in the detection limit of Tf, whereas a 9:1 volume ratio ATPS allows for a 10-fold improvement. (a) Conventional LFA detected Tf at 1 ng/μL, but could not detect Tf at 0.5 ng/μL, leading to false negative results. 1:1 volume ratio ATPS with 3-D paper wells successfully detected Tf at 0.5 ng/μL. (b) Conventional LFA detected Tf at 1 ng/μL, but could not detect Tf at 0.1 ng/μL, leading to false negative results. 9:1 volume ratio ATPS with 3-D paper wells successfully detected Tf at 0.1 ng/μL.

38 depicts an exemplary ATPS-LFA system.

39 depicts a schematic of an LFA sandwich assay. Positive results (top) are represented by two bands in the serum line and control line, while negative results (bottom) are represented by a single band in the control line.

40A- C depict the aqueous biphasic PEG-salt system after incubation for (a) 10 min, (b) 30 min, and (c) 24 h at 37 °C. White paper was filled with the letter “X” to help visualize the turbidity of each phase. Arrows indicate the location of the macroscopic interface.

41 depicts experimentally determined concentration factors of M13 at various time points in PEG/salt ATPS. Error bars represent standard deviation from triplicate measurements.

42 depicts experimentally determined concentration factors of M13 at various volume ratios in PEG/salt ATPS. Symbols correspond to experimental data, and error bars represent standard deviations from triplicate measurements. The solid line corresponds to the prediction from equation (6).

43A-F LFA for detecting M13 without prior concentration step using PEG/salt ATPS. Panel (A) shows the negative control without M13 added. The residual solution contained M13 at concentrations of (b) 1x10 ¹⁰ , (c) 3.2x10 ⁹ , (d) 1x10 ⁹ , (e) 3.2x10 ⁸ , and (f) 1x10 ⁸ pfu/mL.

44A-F: LFA for detecting M13 using a pre-concentration step. Panel (A) shows the negative control without M13 added. The residual solution initially contained M13 at concentrations of (b) 1x10 ⁹ , (c) 3.2x10 ⁸ , (d) 1x10 ⁸ , (e) 3.2x10 ⁷ , and (f) 1x10 ⁷ pfu/mL.

Figure 45 LFA signal intensity for M13 with (■) and without (▲) pre-enrichment step. Error bars represent standard deviation from at least three measurements.

46 depicts a schematic of the ATPS-LFA system in PBS.

47 depicts a schematic representation of competition-based LFA and interpretation of positive and negative results.

Figure 48a-d shows a schematic diagram of ATPS (a) GNPs and surface modifications of GNPs that affect the splitting behavior in the function of each component. To demonstrate that the cleavage behavior of GNPs in the present PEG-salt ATPS can be tailored, various amounts of PEG were conjugated to GNPs to engineer their cleavage behavior: (b) of 5000:1 PEG:GNP during conjugation. Using the molar ratio, the resulting GNP is preferentially cleaved on the PEG-rich phase. (c) Using a molar ratio of 1000:1 PEG:GNP during conjugation, the GNPs are cleaved in the PEG-deficient subphase but aggregated as they have a high salt concentration. These aggregated GNPs could not be used for subsequent detection assays. (d) Using a molar ratio of 3000:1 PEG:GNP during conjugation, the resulting GNP is extremely cleaved at the interface. For (b), (c) and (d), the observed red color at the top of the liquid-air interface is due to reflection and not the presence of nanoprobes.

49 depicts the results of LFA for detection of Tf in PBS without (top panel) and with (bottom panel) a pre-concentration step using a PEG/salt ATPS interfacial extraction step.

50 depicts the results of LFA for detection of Tf in FBS without (upper panel) and with (lower panel) a pre-concentration step using a PEG/salt ATPS interfacial extraction step.

Figure 51 depicts the results of LFA for detection of Tf in synthetic urine without (top panel) and with (bottom panel) a pre-concentration step using a PEG/salt ATPS interfacial extraction step.

52 depicts simultaneous concentration and detection of target analytes using micellar ATPS.

53A-B show a comparison of the time required to achieve phase separation of a 1:9 volume ratio Triton X-114 system in PBS. Brilliant blue FCF dye and cherry BSA-GN were colorimetric indicators for the lower, micelle rich and upper, micelle deficient phases, respectively. (a) At 25 °C, Triton X-114 ATPS achieved macroscopic phase separation equilibrium at 8 h in vitro. (b) When the 3-D paper wick was applied to mixed ATPS, phase separation occurred within the wick at 30 s, as the micellar-rich domain remained near the bottom of the wick and the micellar-poor domain allowed the wick to flow more freely. has already been observed. Upon symbi ejection, the distinct phases remained as separated from each other and the micellar-rich phase remained as concentrated in a small volume in front of the reading, indicating a more complete separation at 180 s (3 min).

54A-B illustrate the incorporation of paper wicks and Triton X-114 micellar ATPS with LFA. (a) The integrated diagnostic strip consists of a detection region containing immobilized test and control line components, followed by a concentrated region where phase separation occurs. (b). A true negative test was confirmed within 20 minutes when a solution containing no pLDH was analyzed.

Figure 55 shows that the paper-based 1:9 volume ratio micellar ATPS achieved a 10-fold improvement in the detection limit of pLDH in PBS at 25 °C. Standard LFA detected pLDH at 10 ng μL ⁻¹ but could not successfully detect pLDH at 1 ng μL ⁻¹ . The integrated diagnostic strip successfully detected pLDH at 1 ng μL ⁻¹ .

Figure 56 shows that paper-based 9:1 volume ratio micellar ATPS achieved a 10-fold improvement in the detection limit of pLDH in undiluted FBS at 25 °C. Standard LFA detected pLDH at 10 ng μL ⁻¹ but could not successfully detect pLDH at 1 ng μL ⁻¹ . The integrated diagnostic strip successfully detected pLDH at 1 ng μL ⁻¹ .

57A-D show (a) PEG 4600 and potassium phosphate on glass fibers, (B) PPG 425 and potassium phosphate on glass fibers, (c) PEG 8000 and dextran 9000 on cellulose and (d) PEG 4600 and Phase separation results using Dextran 35000 are shown.

58A-B show phase separation on different paper forms leading to either (a) a salt phase, a reading fluid, or (b) a PEG phase, a reading fluid.

59A-D depict different designs for dehydrating polymer ATPS components including placing hydrophobic polymers (eg, (a) narrow regions of LFAs, (b) centers of broad regions of LFAs, (c) A region of the LFA extending from a narrow region of the LFA to a broad region of the LFA and (d) mainly PEG to a broad region of the LFA) phase.

60 depicts time course images of phase separation on a dehydrated polymer ATPS-LFA system.

Figure 61 depicts time course images of phase separation on a dehydrated polymer ATPS-LFA system.

62 shows a design in which the dehydrated nanoprobe segments are wider than the width of the detection membrane (the width is defined as the dimension within the flow plane but perpendicular to the flow direction).

63A-B depict successful phase separation in a wide range of design LFAs with (a) no ATPS or (b) ATPS.

64 is an image of an experiment in which flow cannot continue due to the viscosity of the PEG-rich phase of the polymer ATPS, which dramatically slows the flow within the detection membrane.

Figure 65 shows that the PEG rich phase that dramatically slows the flow in the detection membrane can be resolved by leaving a blank spacer that does not contain PEG.

66 depicts a dehydrated LFA design with a constant slope from the source of fluid to the LFA membrane to reduce the amount of nanoprobes left behind.

67 illustrates a dehydrated ATPS-LFA system using PEG/salt ATPS.

68 illustrates a 3D paper well design with a paper pad containing a dehydrated polymeric ATPS component.

69A-B depict separations on 3D paper wells LFA with paper pads in which the wells contain a dehydrated polymeric ATPS component that is either (a) at the start end of the LFA or (b) not present at the start end of the LFA.

70 depicts an exemplary two-well configuration design in which there are two different 3D wells separated by some length of horizontal paper. The first well contains the dehydrated ATPS component. The second well allows more time and space for phase separation.

71 shows that MF1 paper is compatible with blood samples in PEG-salt solutions.

72 shows that filtration of blood samples on MF1 paper can be slow.

73A-B show the results of blood sample and PEG-salt solution phase separation using (a) 3-layer 3-D wells or (b) 5-layer 3-D wells on filter paper.

74 depicts the results of blood sample and PEG-salt solution phase separation using 3-D wells on paper.

Figure 75 depicts the results of blood sample and PEG-salt phase separation using 3-D wells containing layers of filtered and concentrated paper.

76 shows C. in virus transport medium (VTM) using sandwich format LFA . The detection of C. trachomatis is shown .

77 depicts S. using sandwich format LFA and micellar ATPS . The detection of S. mutans is shown .

78 shows S. using sandwich format LFA and PEG/salt ATPS . The detection of mutans is shown.

79 depicts the detection of troponin using competition format LFA and PEG/salt ATPS.

Figure 80a-c shows a schematic illustration (a) and results of adding mixed ATPS on a paper membrane, where it was possible for the two phases of ATPS to completely separate inside the glass just before flowing through the paper. The PEG-deficient phase containing purple dextran-coated gold nanoparticles (empty arrow) was observed to flow rapidly through the paper, whereas the PEG-rich phase containing blue dye (solid arrow) was retained at the beginning of the paper membrane. . The enhanced phase separation that occurred within the paper membrane was evident when using (b) 1:1 volume ratio ATPS or (c) 9:1 volume ratio ATPS.

81 depicts the detection of pLDH in FBS samples at 0.1 ng/mL using 9:1 volumes Triton-X-PBS ATPS and 3-D LFA.

82 shows a schematic illustration of a probe-analyte complex added to LFA in one phase of ATPS after phase separation and concentration of a mixed ATPS solution or one phase for simultaneous concentration and detection. .

details

Provided herein are methods, devices, kits and/or systems that enable implementation at a point of care (POC) or point of use and provide useful tools for medical, consumer and other uses. The tests (assay devices and/or systems) provided herein are rapid, simple, easy to use, and, if any, can be readily performed by individuals with little clinical diagnostic training. The test is very sensitive and reliable. In certain embodiments, the devices and methods approach or meet the unexpectedly surprising sensitivity, e.g., the sensitivity of a laboratory-based assay, such as an enzyme-linked immunosorbent assay (ELISA), compared to the detection limits of previous LFA assays, and in certain cases The concentration capacity of the aqueous multiphase system is used in conjunction with LFA to provide at least about 10-fold to about 100-fold improvement in excess of this.

I. Device

Disclosed herein is a device for detecting and/or quantifying a target analyte in a sample using ( eg, configured to provide) a lateral flow assay (LFA) and an aqueous two-phase system (ATPS), Here, ATPS includes a mixed phase solution separated into a first phase solution and a second phase solution, and LFA includes a test line and a control line for detecting a target analyte. In some embodiments, the separation of the mixed phase into a first phase solution and a second phase solution occurs within the LFA. In some embodiments, the separation of the mixed phase into a first phase solution and a second phase solution does not occur in the LFA. In some embodiments, the first phase solution flows through the LFA more rapidly than the second phase solution and is referred to as the leading fluid, whereas the second phase solution is referred to as a stagnant fluid. In some embodiments, the second phase solution flows through the LFA more rapidly than the first phase solution and is termed a leading fluid, whereas the first phase solution is termed a stagnant fluid. In some embodiments, the target analyte is partitioned/concentrated into a reading fluid. In some embodiments, the target analyte is partitioned/concentrated in the retention fluid.

Configuration and operation

In some embodiments, the device comprises a port for administering ATPS and/or a sample to the device to the device. In some embodiments, the device comprises a first component comprising ATPS and a second component comprising LFA. In some embodiments, the first component and the second component are provided as separate components. In some embodiments, the first component and the second component are joined together by a user. In some embodiments, the first component has a first port connected to the ATPS. In some embodiments, the first component comprises a chamber for ATPS. In some embodiments, the first port is connected to the chamber. In some embodiments, the first port is configured for administering a sample and/or a target analyte to the first component. In some embodiments, the first port is configured for administering ATPS or a component thereof to the first component. In some embodiments, the second port is configured for administering a sample and/or a target analyte to the second component. In some embodiments, the second port is configured for administering ATPS or a component thereof to the second component.

In some embodiments, the device comprises multiple chambers. In some embodiments, the first component comprises a first chamber that holds the sample and/or ATPS solution and allows phase separation to occur. In some embodiments, the second component comprises a first chamber ( eg, a detection chamber) that receives the LFA and facilitates application of the target analyte to the LFA after phase separation has occurred. In some embodiments, additional chambers may be added to facilitate flow, such as using a pressure differential or creating a vacuum. In some embodiments, the device collects target analytes from ATPS and transports them to the LFA using a mechanical component.

In some embodiments, ATPS and LFA are integrated (ATPS-LFA), ie, the ATPS component is coupled to the LFA component either directly or via a connector. In certain embodiments, ATPS and LFA are provided as separate components that are assembled together, eg , by the user, or they are provided as a single integrated unit. In some embodiments, the device comprises a connection between the ATPS and the LFA ( eg , an ATPS-LFA connector). Illustrative, but non-limiting examples of ATPS-LFT connectors include tubes, ports, valves, funnels, gates, pumps, holes, channels, filters, combinations thereof, and the like. In some embodiments, the integrated device comprises a dehydrated ATPS component on LFA. In some embodiments, the device comprises a single port, wherein the single port is connected to the ATPS-LFA. In some embodiments, the single port is configured for administering a sample and/or target analyte to the ATPS-LFA. In some embodiments, the single port is configured for administering ATPS or a component thereof to the ATPS-LFA. In some embodiments, ports include, but are not limited to, structures selected from tubes, funnels, valves, syringes, straws, channels, plungers, pistons, pumps, combinations thereof, and the like.

In some embodiments, the device comprises an LFA strip. In some embodiments, the device is configured for inserting an LFA strip into ATPS. In some embodiments, ATPS and LFA are contained in a single housing.

In some embodiments, the device further comprises an actuator to deliver the sample and/or target analyte to the ATPS. In some embodiments, the device further comprises an actuator to deliver the solution to the LFA.

portability

In some embodiments, the device is a portable device. In some embodiments, the device weighs less than about 100 ounces, less than about 90 ounces, less than about 80 ounces, less than about 70 ounces, less than about 60 ounces, less than about 50 ounces, less than about 40 ounces, less than about 32 ounces, about less than 24 ounces, less than about 16 ounces, less than about 8 ounces, less than about 4 ounces, less than about 2 ounces, or less than about 1 ounce. In some embodiments, a plurality of devices are packaged in a portable container. In some embodiments, the maximum length of the device is about 1 inch, about 2 inches, about 3 inches, about 4 inches, about 6 inches, about 7 inches, about 8 inches, about 9 inches, about 10 inches, about 12 inches, about 14 inches, about 16 inches, about 18 inches, about 19 inches, about 20 inches, about 24 inches, about 26 inches, about 28 inches, or about 30 inches. In some embodiments, the device folds down to a smaller proportion when deployed such that it can be conveniently transported and/or stored.

4 illustrates a portable device capable of combining ATPS with LFA using a piston and plunger system. In one exemplary embodiment, the concentration and detection chambers are connected by a check valve, or similar mechanism to ensure that the LFA strip does not prematurely contact the non-enriched sample. A piston and plunger system may be used to draw the sample into the concentration chamber. Concentration can occur inside the device by premixing the sample with the ATPS solution prior to analyte extraction prior to sampling into the device, or concentration can occur inside the device by preloading the ATPS solution as a liquid or in dehydrated form inside the concentration chamber. After phase separation has occurred, the user can initiate the detection process using the piston/plunger to re-direct a predetermined volume of the concentrated analyte from the concentration chamber through the check valve to the detection chamber. The position of the check valve can be modified for transport to the LFA strip in the desired bulk phase (upper or lower phase) in which the analyte is present.

5 illustrates a device design demonstrating collection on the upper bulk for application to an LFA strip. In the illustrated embodiment, the sample premixed with the ATPS solution is withdrawn into the device. After phase separation, the user can initiate the detection phase by pressing a button to bring the LFA test strip(s) into contact with the analyte concentrated on top of ATPS. In various embodiments, the ATPS solution component may be provided preloaded to the device in liquid or dehydrated form.

6 provides an illustrative, but non-limiting example of a portable cassette device. The sample and ATPS solution may first be loaded into the sample wells on the cassette device. Wells can be designed as detachable components that can be attached to the cassette after loading the sample. After phase separation has occurred, the detection step can be initiated by mechanical displacement of the LFA test strips or sample wells.

7 illustrates one configuration in which a magnetic capture probe can be applied for integration of ATPS and LFA in a portable device. Similar to the device presented in FIG. 4 , the illustrated device may use a plunger and piston system to transport the concentrated analyte collected by an external magnet to the detection chamber. In some embodiments, the device does not require a power source ( eg , electricity).

detection time

In some embodiments, the device has a detection time. In some embodiments, the detection time includes a phase separation time. In some embodiments, the detection time comprises a flow time.

In some embodiments, the detection time ( eg , the time between application of the sample to the device and detection of the target analyte in a control/test, etc.) is less than about 2 hours, less than about 1.5 hours, less than about 1 hour, or less than about half an hour. In some embodiments, the detection time is less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, the detection time is less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute. In some embodiments, the detection time is less than about 30 seconds, less than about 20 seconds, or less than about 10 seconds.

In some embodiments, the phase separation time ( eg , the time it takes for the first/second phase solution to separate from the mixed phase solution of ATPS) is less than about 2 hours, less than about 1.5 hours, less than about 1 hour, or less than about 0.5 hours. am. In some embodiments, the detection time is less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, the phase separation time is less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute. In some embodiments, the phase separation time is less than about 30 seconds, less than about 20 seconds, or less than about 10 seconds.

In some embodiments, the flow time ( eg , the time it takes for a solution containing a target analyte to travel from the sample pad to the test line and control line of the LFA) is less than about 2 hours, less than about 1.5 hours, less than about 1 hour, or less than about 0.5 hours. In some embodiments, the flow time is less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, the detection time is less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute. In some embodiments, the flow time is less than about 30 seconds, less than about 20 seconds, or less than about 10 seconds.

detection limit

In some embodiments, the device has a detection limit, wherein the detection limit is the minimum amount of target analyte that must be present in the sample to be detected.

In some embodiments, the minimum amount of target analyte in the sample is about 0.01 ng/ml, about 0.05 ng/ml, about 0.1 ng/ml, about 0.15 ng/ml, about 0.20 ng/ml, about 0.25 ng/ml, about 0.3 ng/ml, about 0.35 ng/ml, about 0.40 ng/ml, about 0.45 ng/ml, about 0.5 ng/ml, about 0.55 ng/ml, about 0.60 ng/ml, about 0.65 ng/ml, about 0.7 ng/ ml, about 0.75 ng/ml, about 0.80 ng/ml, about 0.85 ng/ml, about 0.9 ng/ml, about 0.95 ng/ml, or about 1 ng/ml. In some embodiments, the minimum amount of target analyte in the sample is about 1 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, about 120 ng/ml, about 130 ng/ml, about 140 ng/ml ml, about 150 ng/ml, about 160 μg/ml, about 170 ng/ml, about 180 ng/ml, about 190 ng/ml, about 200 ng/ml, about 250 ng/ml, about 300 ng/ml, About 350 ng/ml, about 400 ng/ml, about 450 ng/ml, about 500 ng/ml, about 550 ng/ml, about 600 ng/ml, about 650 ng/ml, about 700 ng/ml, about 750 ng/ml, about 800 ng/ml, about 850 ng/ml, about 900 ng/ml, about 980 ng/ml or about 1000 ng/ml.

In some embodiments, the minimum amount of analyte is about 1 μg/ml, about 2 μg/ml, about 3 μg/ml, about 4 μg/ml, about 5 μg/ml, about 6 μg/ml, about 7 μg/ml , about 8 μg/ml, about 9 μg/ml, about 10 μg/ml, about 11 μg/ml, about 12 μg/ml, about 13 μg/ml, about 14 μg/ml, about 15 μg/ml, about 16 μg/ml, about 17 μg/ml, about 18 μg/ml, about 19 μg/ml or about 20 μg/ml. In some embodiments, the minimum amount of target analyte is about 10 μg/ml, about 20 μg/ml, about 30 μg/ml, about 40 μg/ml, about 50 μg/ml, about 60 μg/ml, about 70 μg/ml ml, about 80 μg/ml, about 90 μg/ml or about 100 μg/ml. In some embodiments, the minimum amount of target analyte is about 10 μg/ml.

In some embodiments, the minimum amount of target analyte is about 1 part per trillion, about 10 parts per trillion, or about 100 parts per trillion. In some embodiments, the minimum amount of target analyte is about 1 part per billion, about 10 parts per billion, about 100 parts per billion, about 1000 parts per billion, about 10,000 parts per billion, or about 100,000 parts per billion. In some embodiments, the minimum amount of target analyte is about 1 part per thousand, about 10 parts per thousand, or about 100 parts per thousand.

In some embodiments, the target analyte is present on or derived from a cell. In some embodiments, the sample has a minimal concentration of cells to be present on or derived from the cell in which the target analyte is detected. In some embodiments, the minimum concentration of cells is about 1 cell/ml sample, about 10 cells/ml sample, about 20 cells/ml sample, about 30 cells/ml sample, about 40 cells/ml sample, about 50 cells/ml sample. cells, about 60 cells per ml of sample, about 70 cells per ml of sample, about 80 cells per ml of sample, about 90 cells per ml of sample or about 100 cells per ml of sample.

In some embodiments, the minimum concentration of cells is about 1x10 ⁰ cells/mL, about 1x10 ¹ cells/mL, about 1x10 ² cells/mL, about 1x10 ³ cells/mL, about 1x10 ⁴ cells/mL, about 1x10 ⁵ cells/mL mL, about 1× ^{10 6} cells/mL, about 1× ^{10 7} cells/mL, about 1× ^{10 8} cells/mL, or about 1× ^{10 9} cells/mL. In some embodiments, the minimum concentration of cells is about 1× ^{10 6} cells/mL.

In some embodiments, the target analyte is a viral particle and is present on or derived from a virus. In some embodiments, the sample has a minimal concentration of viral particles such that the target analyte is present on or derived from the virus being detected. In some embodiments, the minimum concentration of viral particles in the sample is about 1x10 ⁰ pfu/mL, about 1x10 ¹ pfu/mL, about 1x10 ² pfu/mL, about 1x10 ³ pfu/mL, about 1x10 ⁴ pfu/mL, about 1x10 ⁵ pfu/mL, about 1x10 ⁶ pfu/mL, about 1x10 ⁷ pfu/mL, about 1x10 ⁸ pfu/mL, about 1x10 ⁹ pfu/mL, about 1x10 ¹⁰ pfu/mL, about 1x10 ¹¹ pfu/mL, or about 1x10 ¹² pfu/mL.

In some embodiments, the minimum concentration of viral particles in the sample is about 1x10 ⁶ pfu/mL, 2x10 ⁶ pfu/mL, 3x10 ⁶ pfu/mL, 4x10 ⁶ pfu/mL, 5x10 ⁶ pfu/mL, 6x10 ⁶ pfu/mL, 7x10 ⁶ pfu/mL, 8x10 ⁶ pfu/mL, 9x10 ⁶ pfu/mL, about 1x10 ⁷ pfu/mL, about 2x10 ⁷ pfu/mL, about 3x10 ⁷ pfu/mL, about 4x10 ⁷ pfu/mL, about 5x10 ⁷ pfu /mL, about 6x10 ⁷ pfu/mL, about 7x10 ⁷ pfu/mL, about 8x10 ⁷ pfu/mL, about 9x10 ⁷ pfu/mL, about 1x10 ⁸ pfu/mL, about 2x10 ⁸ pfu/mL, about 3x10 ⁸ pfu/mL mL, about 4x10 ⁸ pfu/mL, about 5x10 ⁸ pfu/mL, about 6x10 ⁸ pfu/mL, about 7x10 ⁸ pfu/mL, about 8x10 ⁸ pfu/mL, about 9x10 ⁸ pfu/mL, about 1x10 ⁹ pfu/mL , about 2x10 ⁹ pfu/mL, about 3x10 ⁹ pfu/mL, about 4x10 ⁹ pfu/mL, about 5x10 ⁹ pfu/mL, about 6x10 ⁹ pfu/mL, about 7x10 ⁹ pfu/mL, about 8x10 ⁹ pfu/mL, About 9x10 ⁹ pfu/mL, about 1x10 ¹⁰ pfu/mL, about 2x10 ¹⁰ pfu/mL, about 3x10 ¹⁰ pfu/mL, about 4x10 ¹⁰ pfu/mL, about 5x10 ¹⁰ pfu/mL, about 6x10 ¹⁰ pfu/mL, about 7×10 ¹⁰ pfu/mL, about 8× ^{10 10} pfu/mL, about 9×10 ¹⁰ pfu/mL or about 1× ^{10 11} pfu/mL. In some embodiments, the minimum concentration of viral particles in a sample is about 1× ^{10 7} pfu/mL. In some embodiments, the minimum concentration of viral particles in a sample is about 1× ^{10 8} pfu/mL. In some embodiments, the minimum concentration of viral particles in a sample is about 1× ^{10 9} pfu/mL.

temperature

In some embodiments, of course the temperature must be below the combustion/melting/flash point of the device material and below the temperature at which the components of the device volatilize and sublimate or break down and above the freezing point of the components of the device when performing the assay, but Works regardless of temperature. In some embodiments, the device operates at room temperature. In some embodiments, the device operates at a temperature between about -50°C and about 60°C. In some embodiments, the device operates at a temperature between about -10°C and about 45°C. In some embodiments, the device operates at a temperature between about 10°C and about 30°C.

aqueous two-phase system ( ATPS)

In certain embodiments, the device is configured to support aqueous two-phase system (ATPS) methods and assays using such devices provided herein. In some embodiments, ATPS comprises a phase solution. The term “phase solution” generally refers to a first phase solution or a second phase solution of ATPS. In some embodiments, a phase solution is present as a mixed solution ( eg, including a first/second phase solution). In some embodiments, the phase solution is the first/second phase solution after it has been resolved from the mixed solution of ATPS. In some embodiments, the phase solution is the first/second phase solution after it has been resolved from the mixed solution in LFA. This may refer to a second phase solution while it is in a mixed state ( eg, including a first phase solution). In some embodiments, the phase solution is a reading fluid in LFA. In some embodiments, the phase solution is a retention fluid in LFA.

In some embodiments, ATPS comprises two aqueous solutions that are initially mixed, a first phase solution and a second phase solution ( eg , a mixed phase solution). In some embodiments, the mixed phase solution is a homogeneous solution. In some embodiments, the first phase solution and the second phase solution are immiscible. In some embodiments, immiscibility is driven by a change in temperature and/or a change in the concentration of different components, such as salts. In some embodiments, the first/second phase solutions include components such as micelles, salts and/or polymers. In some embodiments, the target analyte contacted with ATPS is preferentially partitioned and partitioned into the first phase solution over the second phase solution based on its physical and chemical properties, such as size, shape, hydrophobicity and charge, and / or concentrated, or vice versa. In some embodiments, a target analyte ( eg , a component of a mammalian cell, bacterium, or virus) is primarily (or extremely) partitioned into a first or second phase solution of ATPS, and thus concentrated in ATPS. In some embodiments, the target analyte is concentrated by adjusting the volume ratio between the first phase solution and the second phase solution. In some embodiments, the target analyte is enriched by reducing the volume of the phase into which the analyte is resolved. For example, in some embodiments, the target analyte has a 1:9 volume ratio of the first phase solution to the second phase solution, e.g. , because the volume of the phase into which the analyte is extremely divided is 1/10 of the total volume. Concentrate up to 10 times in the first phase solution by using

In some embodiments, different concentrations are obtained by using different ratios. Thus, in some embodiments, the ratio of the first phase solution to the second phase solution is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 and about 1:10. In some embodiments, the ratio of the first phase solution to the second phase solution is about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1: 80, about 1:90 and about 1:100. In some embodiments, the ratio of the first phase solution to the second phase solution is about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1: 800, about 1:900 and about 1:1000.

In some embodiments, the ratio of the second phase solution to the first phase solution is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1: 7, about 1:8, about 1:9 and about 1:10. In some embodiments, the ratio of the second phase solution to the first phase solution is about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1: 80, about 1:90 and about 1:100. In some embodiments, the ratio of the second phase solution to the first phase solution is about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1: 800, about 1:900 and about 1:1000.

In some embodiments, the analyte is partitioned substantially uniformly between the first phase solution and the second phase solution to prevent concentration of the analyte. In such systems, enrichment of the target analyte is achieved by introducing an additional component, eg, a probe that captures the target analyte, wherein the probe is partitioned primarily into one phase to allow for the cleavage behavior of the target analyte to enable enrichment. to improve In some embodiments, the first/second phase solutions containing the concentrated analyte are collected and subjected to LFA. The use of these additional components is not limited to a substantially uniform partitioning system. Even where the analyte is primarily partitioned into one phase, it is understood that, in some embodiments, this partitioning is enhanced by the use of additional components ( eg probes) as described above.

In some embodiments, the first/second phase solution comprises a micellar solution. In some embodiments, the micellar solution comprises a nonionic surfactant. In some embodiments, the micellar solution comprises a detergent. In some embodiments, the micellar solution comprises Triton-X. In some embodiments, the micellar solution comprises a Triton-X-like polymer such as, but not limited to, Igepal CA-630 and Nonidet P-40. In some embodiments, the micellar solution consists essentially of Triton-X.

In some embodiments, the micellar solution is from about 0.01 centipoise to about 5000 centipoise, from about 0.01 centipoise to about 4500 centipoise, from about 0.01 centipoise to about 4000 centipoise, from about 0.01 centipoise to about 3500 centipoise, about 0.01 centipoise to about 3000 centipoise, about 0.01 centipoise to about 2500 centipoise, about 0.01 centipoise to about 2000 centipoise, about 0.01 centipoise to about 1500 It has a viscosity (at room temperature, about 25° C.) of centipoise, about 0.01 centipoise to about 1000 centipoise, or about 0.01 centipoise to about 500 centipoise. In some embodiments, the micellar solution is from about 0.01 centipoise to about 450 centipoise, from about 0.01 centipoise to about 400 centipoise, from about 0.01 centipoise to about 350 centipoise, from about 0.01 centipoise to about 300 centipoise, about 0.01 centipoise to about 250 centipoise, about 0.01 centipoise to about 200 centipoise, about 0.01 centipoise to about 150 centipoise, or about 0.01 centipoise to about 100 It has a viscosity of centipoise (at room temperature, about 25° C.).

In some embodiments, the first/second phase solution comprises a polymer ( eg , a polymer solution). In certain embodiments, the polymer is polyethylene glycol (PEG). In various embodiments, the PEG may have a molecular weight between 1000 and 100,000. In certain embodiments, the PEG is selected from PEG-4600, PEG-8000 and PEG-20,000, PEG. In certain embodiments, the polymer is polypropylene glycol (PPG). In various embodiments, the PPG may have a molecular weight between 100 and 10,000. In certain embodiments, the PPG is selected from PPG 425. In certain embodiments, the polymer is dextran. In various embodiments, the dextran can have a molecular weight between 1000 and 1,000,000. In certain embodiments, the dextran is selected from dextran 6000, dextran 9000, dextran-35,000 and dextran-200,000.

In some embodiments, the polymer solution comprises about 0.01% w/w, about 0.05% w/w, about 0.1% w/w, about 0.15% w/w, about 0.2% w/w, about 0.25% w/w, about 0.3% w/w, about 0.35% w/w, about 0.4% w/w, about 0.45% w/w, about 0.5% w/w, about 0.55% w/w, about 0.6% w/w, about 0.65% w/w, about 0.7% w/w, about 0.75% w/w, about 0.8% w/w, about 0.85% w/w, about 0.9% w/w, about 0.95% w/w, or about 1 % w/w polymer solutions. In some embodiments, the polymer solution comprises about 1% w/w, about 2% w/w, about 3% w/w, about 4% w/w, about 5% w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, about 20% w/w, about 21 % w/w, about 22% w/w, about 23% w/w, about 24% w/w, about 25% w/w, about 26% w/w, about 27% w/w, about 28% w/w, about 29% w/w, about 30% w/w, about 31% w/w, about 32% w/w, about 33% w/w, about 34% w/w, about 35% w /w, about 36% w/w, about 37% w/w, about 38% w/w, about 39% w/w, about 40% w/w, about 41% w/w, about 42% w/w w, about 43% w/w, about 44% w/w, about 45% w/w, about 46% w/w, about 47% w/w, about 48% w/w, about 49% w/w and about 50% w/w of a polymer solution. In some embodiments, the polymer solution comprises about 10% w/w, about 20% w/w, about 30% w/w, about 40% w/w, about 50% w/w, about 60% w/w, about 70% w/w, about 80% w/w or about 90% w/w of a polymer solution. In some embodiments, the polymer solution is selected from about 10% w/w to about 80% w/w polymer solution. In some embodiments, the polymer solution is selected from about 10% w/w to about 25% w/w polymer solution.

In some embodiments, the first/second phase solution comprises a salt ( eg , the first/second phase solution is a salt solution). In some embodiments, the target analyte and/or probe-analyte complex is resolved into a salt solution. In some embodiments, the target analyte and/or probe-analyte complex is resolved into a salt solution, wherein the salt solution comprises a cosmotropic salt. In some embodiments, the target analyte and/or probe-analyte complex is resolved into a salt solution, wherein the salt solution comprises a chaotropic salt. In some embodiments, the salt is selected from a magnesium salt, a lithium salt, a sodium salt, a potassium salt, a cesium salt, a zinc salt and an aluminum salt. In some embodiments, the salt is selected from a bromide salt, an iodide salt, a fluoride salt, a carbonate salt, a sulfate salt, a citrate salt, a carboxylate salt, a borate salt, and a phosphate salt. In some embodiments, the salt is potassium phosphate. In some embodiments, the salt is aluminum sulfate.

In some embodiments, the salt solution comprises about 0.01% w/w, about 0.05% w/w, about 0.1% w/w, about 0.15% w/w, about 0.2% w/w, about 0.25% w/w, about 0.3% w/w, about 0.35% w/w, about 0.4% w/w, about 0.45% w/w, about 0.5% w/w, about 0.55% w/w, about 0.6% w/w, about 0.65% w/w, about 0.7% w/w, about 0.75% w/w, about 0.8% w/w, about 0.85% w/w, about 0.9% w/w, about 0.95% w/w, or about 1 % w/w salt solutions. In some embodiments, the salt solution comprises about 1% w/w, about 2% w/w, about 3% w/w, about 4% w/w, about 5% w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, about 20% w/w, about 21 % w/w, about 22% w/w, about 23% w/w, about 24% w/w, about 25% w/w, about 26% w/w, about 27% w/w, about 28% w/w, about 29% w/w, about 30% w/w, about 31% w/w, about 32% w/w, about 33% w/w, about 34% w/w, about 35% w /w, about 36% w/w, about 37% w/w, about 38% w/w, about 39% w/w, about 40% w/w, about 41% w/w, about 42% w/w w, about 43% w/w, about 44% w/w, about 45% w/w, about 46% w/w, about 47% w/w, about 48% w/w, about 49% w/w and about 50% w/w of a salt solution. In some embodiments, the salt solution is selected from about 0.1% w/w to about 10% salt solution. In some embodiments, the salt solution is selected from about 1% w/w to about 10% salt solution.

In some embodiments, the first/second phase solutions comprise a solvent that is immiscible with water. In some embodiments, the solvent comprises a non-polar organic solvent. In some embodiments, the solvent comprises an oil. In some embodiments, the solvent is selected from pentane, cyclopentane, benzene, 1,4-dioxane, diethyl ether, dichloromethane, chloroform, toluene and hexane.

In some embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a polymer. In some embodiments, the second phase solution comprises a micellar solution and the first phase solution comprises a polymer. In some embodiments, the first phase solution comprises a micellar solution and the second phase solution comprises a salt. In some embodiments, the second phase solution comprises a micellar solution and the first phase solution comprises a salt. In some embodiments, the micellar solution is a Triton-X solution. In some embodiments, the first phase solution comprises a first polymer and the second phase solution comprises a second polymer. In some embodiments, the first/second polymer is selected from polyethylene glycol and dextran. In some embodiments, the first phase solution comprises a polymer and the second phase solution comprises a salt. In some embodiments, the second phase solution comprises a polymer and the first phase solution comprises a salt. In some embodiments, the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate. In some embodiments, the second phase solution comprises polyethylene glycol and the first phase solution comprises potassium phosphate. In some embodiments, the first phase solution comprises a salt and the second phase solution comprises a salt. In some embodiments, the first phase solution comprises a cosmotropic salt and the second phase solution comprises a chaotropic salt. In some embodiments, the second phase solution comprises a cosmotropic salt and the first phase solution comprises a chaotropic salt.

In some embodiments, the first phase solution is selected from component 1 of Table 1 and the second phase solution is selected from component 2 of Table 1. In some embodiments, the second phase solution is selected from component 1 of Table 1, and the second phase solution is selected from component 2 of Table 1.

In some embodiments, the components of Table 1 are suspended or dissolved in a buffer. In some embodiments, the components of Table 1 are suspended/dissolved in a buffer suitable for the biological system from which the sample was derived. In some embodiments, the ingredients of Table 1 are suspended/dissolved in physiological saline solution. In some embodiments, the components of Table 1 are suspended/dissolved in PBS. In some embodiments, the ingredients of Table 1 are suspended/dissolved in water.

Table 1. Exemplary aqueous phase extraction systems

In some embodiments, the device further comprises a collector configured to be placed in contact with the ATPS, wherein the target analyte is partitioned at an interface of the collector and the first phase solution and/or the second phase solution. In some embodiments, the collector comprises a material selected from plastics, mesoporous materials, silica, polypropylene, magnets, magnetic particles, paramagnetic particles, porous materials, grooved materials, and any combination thereof. In some embodiments, the collector comprises polypropylene. In some embodiments, the collector is optimized to increase target analyte collection. In some embodiments, the collector includes pores to maximize surface area. In some embodiments, the width of the pores is about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm or about 100 μm. In some embodiments, the width of the pores is about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1 mm. In some embodiments, the depth of the pores is about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm or about 100 μm. In some embodiments, the depth of the pores is about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1 mm.

Lateral Flow Assay (LFA)

In certain embodiments, the devices and systems described herein are configured to provide a lateral flow assay (LFA) for detecting a target analyte in a sample, wherein the LFA is used in conjunction with an aqueous two-phase system (ATPS). . In some embodiments, the LFA comprises a porous matrix in which ATPS or a component thereof is disposed, wherein the porous matrix is configured to allow ATPS or a component thereof to flow through the porous matrix when the ATPS or component thereof is present in a fluid phase. or has sufficient porosity to permit flow. Such porous LFA devices are referred to herein as paper or paper fluid devices, and these terms are used interchangeably. As noted above, the paper used herein is, in certain embodiments, contemplated for use in the LFA apparatus described herein, but is not limited to sheeting from wood pulp or other fibrous plant material.

In some embodiments, the porous matrix is sufficiently porous to allow the mixed phase solution of ATPS, the first phase solution and/or the second phase solution, and/or the target analyte to flow through the LFA. In some embodiments, the porous matrix is long enough and/or deep enough for the mixed phase solution, the first phase solution, and/or the second phase solution, and/or the target analyte to flow vertically and/or horizontally through the LFA. . In some embodiments, the first phase solution flows through the porous matrix at a first rate and the second phase solution flows through the porous matrix at a second rate, wherein the first rate and the second rate are different. In some embodiments, the LFA porous matrix comprises a material selected from sintered glass ceramics, minerals, cellulose, glass fibers, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and combinations thereof. .

concentrate by flow

Prior phase separation in ATPS was performed under stagnant conditions. However, the inventors have recently discovered that ATPS can phase separate when the solution flows through a paper we have termed a "flow-to-flow concentrate". This finding was interesting and amusing because the paper was found to significantly accelerate the thickening process. Based on this important phenomenon, we designed a paper fluid device to fully integrate the necessary components for combined ATPS and LFA diagnostics. Investigating ATPS as it flows through different types of paper under different conditions has been important in enabling the development of more complex paper fluid detection devices. From our experimental data using different ATPSs, such as polymer-salt ATPS and micellar ATPS, we show that when a uniform ATPS solution is applied to a specific paper material, phase separation and analyte concentration occur when flowing the solution. Able to know. We also demonstrated that this phenomenon is conserved even when preparing ATPS with variable volume ratios, eg, the volume of the upper phase divided by the volume of the lower phase ( see, eg, FIG. 12 ).

In some embodiments, the LFA comprises paper. In some embodiments, the paper comprises a sheet of porous material that allows a fluid to flow therethrough. In some embodiments, the paper comprises a plurality of sheets of porous material that allows a fluid to flow therethrough. In some embodiments, the paper comprises a material selected from cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, charge modified nylon, polyether sulfone, and the like. In some embodiments, the paper is a HI-FLOW PLUS® membrane.

In some embodiments, the paper is a woven paper. In some embodiments, the paper is Whatman paper. In some embodiments, Whatman Z is selected from Whatman S17, Whatman MF1, Whatman VF1, Whatman Fusion 5, Whatman GF/DVA, Whatman LF1, Whatman CF1 and Whatman CF4.

In some embodiments, the paper concentrates the target analyte as it flows through the LFA ( eg , a 'concentrate-by-flow'-based device). In some embodiments, the paper concentrates the target analyte as it flows horizontally through the LFA. In some embodiments, the paper concentrates the target analyte as it flows vertically through the LFA. In some embodiments, the paper concentrates the target analyte as it flows vertically through the LFA due to gravity. In some embodiments, the paper concentrates the target analyte as it flows vertically through the LFA due to capillary action. In some embodiments, the paper has properties that affect what phase solution becomes a “leading fluid”. As a non-limiting example, when using PEG-salt ATPS, adding the solution to the glass fiber paper causes the salt phase to become the reading solution, while using cellulosic paper causes the PEG phase to become the reading solution. In some embodiments, phase separation in paper accelerates phase separation. Also, as a non-limiting example, micellar ATPS typically take hours to separate phases in stationary ATPS, but when applied to paper strips, this phase separation occurs in minutes. This accelerates the diagnostic process by making ATPS, which has traditionally been the rate-determining step in the process, a more realistic option for this rapid paper diagnostic assay. In some embodiments, the 'concentrate along flow' device comprises a PEG-salt ATPS. In some embodiments, the 'concentrate along flow' device comprises micellar ATPS. In some embodiments, the LFA of the 'concentrate along flow' device comprises glass fiber paper. In some embodiments, the LFA of the 'concentrate along flow' device comprises nitrocellulose paper.

In some embodiments, the LFA comprises: a filter to remove blood cells or other particles; A sample pad in which a sample containing a target analyte is applied to the LFA, a detection area ( eg , test line and control line) where the target analyte binds and is detected, and an absorbent pad to absorb excess sample and/or solution applied to the LFA. ( eg dry destination) ( see, eg, FIG. 1 top). In some embodiments, a control line and/or a test line is not itself a line but a region or spot.

In some embodiments, the LFA comprises an LFA strip. The terms “LFA” and “LFA strip” are used interchangeably herein. In some embodiments, the LFA strip has a length greater than its width and depth. In some embodiments, the LFA is rectangular. In some embodiments, the LFA has a shape selected from the group consisting of circles, ovals, squares, polygons, and irregular shapes. In some embodiments, the LFA comprises a plurality of routes and/or junctions. In some embodiments, the LFA strip comprises a sample pad, a detection area, and an absorbent pad. In some embodiments, the detection area is disposed between the sample and the absorbent pad, the absorbent pad wicks the sample from the sample pad towards the detection area to the target analyte.

sandwich black

In some embodiments, the LFA is configured to provide or perform a sandwich assay ( see , eg , FIG. 1 , lower left). In some embodiments, a sandwich assay comprises a capture moiety that binds a target analyte. In some embodiments, the device comprises a probe. In some embodiments, a probe comprises a detectable property (colorimetric, fluorescent, radioactive , etc. ). In some embodiments, a probe comprises a binding moiety ( eg , an antibody) that interacts with a target analyte. In some embodiments, a probe is added to a sample and binds to a target analyte to form a probe-analyte complex. In some embodiments, the probe-analyte complex is applied to a sample pad and flows through the LFA through the absorbent pad. In some embodiments, the target analyte of the probe-analyte complex binds to a capture moiety. In some embodiments, the capture moiety is immobilized in the test line and the probe-analyte complex is immobilized in the test line. In some embodiments, the probe is colorimetric and the test line exhibits a strong color ( eg , a detectable signal) when the probe-analyte complex accumulates in the test line, indicating a positive result. In some embodiments, no target analyte is present in the sample, the probe of the probe-analyte complex does not interact with the capture moiety, and the absence of the test line indicates a negative result. In some embodiments, the LFA comprises a probe capture moiety on a control line that directly interacts with the probe and/or binding moiety, so that, regardless of the presence of the target analyte in the sample, the probe/binding moiety binds to the probe capture moiety. and accumulate above the control line. In some embodiments, the probe capture moiety is a secondary antibody that binds a binding moiety, wherein the binding moiety is a primary antibody that binds a target analyte. In some embodiments, the probe is immobilized and detected in a control line to indicate an efficacy test. In some embodiments, a positive result ( eg , the target analyte is present in the sample) is indicated by a detectable signal in the test line and the control line. In some embodiments, a negative result is indicated by a detectable signal in the control line.

competition test

In some embodiments, the LFA is configured to provide a competition assay (see , eg , FIG. 1 , lower right). In some embodiments, a probe is added to a sample and binds to a target analyte to form a probe-analyte complex. In some embodiments, the LFA comprises a target analyte immobilized in a test line. In some embodiments, the probe is saturated by the target analyte in the sample and the probe does not bind to the target analyte immobilized in the test line. In some embodiments, the absence of a detectable signal on the serum line indicates a positive result. In some embodiments, no target analyte is present in the sample and the probe binds to the target analyte on the test line, resulting in a negative result. In some embodiments, the LFA comprises a probe capture moiety in a control line that directly interacts with the probe, and regardless of the presence of the target analyte in the sample, the probe binds to the probe capture moiety and accumulates in the control line. In some embodiments, the probe is immobilized and detected in a control line to indicate an efficacy test. In some embodiments, a positive result ( eg , a target analyte is present in the sample) is indicated by a signal that is not detectable in the test line, and by a signal that is cloakable in a control line. In some embodiments, a negative result is indicated by a detectable signal in both the test line and the control line.

well

In some embodiments, a sample pad comprises a well. In some embodiments, a well is sufficient to contain a solution selected from the group consisting of at least a portion of ATPS, at least a portion of a first phase solution, at least a portion of a second phase solution, a resuspended solution of a target analyte, and combinations thereof. have a volume

In certain embodiments, the volume of the well ranges from about 1 μL to about 10 μL. In certain embodiments, the volume of the well ranges from about 1 μL to about 100 μL. In certain embodiments, the volume of the well ranges from about 1 μL to about 1000 μL. In certain embodiments, the volume of the well ranges from about 1 μL to about 5000 μL. In certain embodiments, the volume of the well ranges from about 1 mL to about 10 mL. In certain embodiments, the volume of the well ranges from about 1 mL to about 100 mL. In certain embodiments, the volume of the well ranges from about 1 mL to about 1000 mL.

In some embodiments, the resuspended solution comprises a buffer to resuspend the concentrated/extracted target analyte from ATPS.

In some embodiments, the well is disposed at a location on the LFA selected from corner, distal, central, junction, off-center and bend of the LFA. In some embodiments, a well comprises one or more pads selected from salt pads, probe pads, polymer pads, and combinations thereof. In some embodiments, a well comprises a plurality of pads. In some embodiments, the first/second phase solution is separated and/or the target analyte is concentrated as it flows through the plurality of pads. In some embodiments, the first/second phase solutions are separated and/or the target analyte is concentrated as it flows vertically through the plurality of pads. In some embodiments, the first/second phase solutions are separated and/or the target analyte is concentrated as it flows vertically through the plurality of pads due to gravity. In some embodiments, the first/second phase solutions are separated and/or the target analyte is concentrated as it flows vertically through the plurality of pads due to capillary action. In some embodiments, the well is a paper well. In some embodiments, the paper well is a three-dimensional paper structure that holds a larger volume of sample compared to a typical paper strip used in LFA. In some embodiments, the paper well is composed of a paper material in which phase separation occurs and allows subsequent analyte concentration in the reading fluid. In some embodiments, the flow of reading fluid is directed towards an absorbent pad that enables analyte detection ( see, eg , FIG. 13 ).

In some embodiments, the device uses a “concentrate as flow” mechanism while further facilitating macroscopic phase separation and flow using gravity in the wells. In some embodiments, the wells are sufficient to promote phase separation because the first phase solution and the second phase solution may flow at different rates due to differences in the viscosity of the phase solutions as well as differences in affinity for the paper material. provides a cross-sectional area. In some embodiments, a well enhances or facilitates phase separation and/or concentration of the target analyte as the phase solution(s) migrates through the well and appears in the reading fluid. In some embodiments, the LFA test strip is directly connected to the well at a downstream location, so that the analyte concentrated in the reading fluid is first contacted with the LFA strip, and the detection step occurs concurrently with the concentration process to further reduce the total assay time. Reduce.

In some embodiments, the LFA comprises a plurality of wells. In some embodiments, the LFA comprises mixed wells. In some embodiments, LFAs comprise enrichment wells. In some embodiments, ATPS is applied to enrichment wells where phase separation and/or enrichment of the target analyte occurs. In some embodiments, the concentrated analyte is removed from the wells after phase separation and applied to the enriched wells. In some embodiments, the enrichment wells comprise a working buffer that enhances and/or promotes phase separation in the enrichment wells and/or LFA (see, eg, FIG. 14 ).

In some embodiments, the device comprises an actuator for releasing the contents of the well into and/or on the porous matrix. In some embodiments, the actuator comprises a mechanism for puncturing the well.

LFA dehydrated in ATPS

In some embodiments, ATPS or a component thereof is dehydrated on and/or in at least the first portion of the porous matrix. In some embodiments, application of the sample to the device hydrates ATPS, converting ATPS or a component thereof into a fluid phase. Dehydration can make the device more user-friendly when the user only needs to add a sample ( eg saliva) to the device. In some embodiments, a user only has to apply a solution of a sample to a stream to detect the presence/absence of a target analyte. In some embodiments, a solution of the sample flows through the LFA and ATPS is re-solubilized, resulting in phase separation within the LFA and subsequent concentration of the target analyte ( see, eg, FIG. 15 ).

In some embodiments, all components required for a given ATPS are mixed to form a uniform solution, applied to a paper, and then dehydrated. When the sample solution is added to the dehydrated paper strip, the ATPS component rehydrates as the sample flows, leading to phase separation. In some ATPS where the phase containing the concentrated analyte is less viscous and potentially contains less polymer or micelles, the phase flows more rapidly, the concentrated analyte appears in the reading fluid, and is sent to the LFA to initiate detection. reach Additionally, the dehydrated ATPS component segment length and concentration can be adjusted for different applications.

In some embodiments, ATPS is dehydrated over LFA. In some embodiments, the first ATPS component is dehydrated on LFA. In some embodiments, the second ATPS component dehydrates the LFA. In some embodiments, the mixed phase solution is dehydrated over LFA. In some embodiments, the first phase solution component and/or the first ATPS component are dehydrated over the first portion of the LFA. In some embodiments, the second phase solution component and/or the second ATPS component are dehydrated over the second portion of the LFA. In some embodiments, the first portion and the second portion are the same. In some embodiments, the first portion and the second portion are different. As a non-limiting example, in PEG-salt ATPS, PEG and salt solution are dehydrated separately in different paper parts or segments ( see, eg, FIG. 16 ). In some embodiments, dehydration of the first/second phase solution and/or ATPS component over different portions of the LFA provides a more uniform concentration of the first/second phase solution component or ATPS component. In some embodiments, dehydration of the first/second phase solution component and/or ATPS component over different portions causes the first phase solution or ATPS component to flow in a first direction after hydration and the second phase solution and/or ATPS component to allow to flow in a second flow direction after hydration, wherein the first direction and the second direction are different. In some embodiments, the target product is concentrated in the first direction and not in the second direction. In some embodiments, the target analyte is enriched in the second direction rather than in the first direction. In some embodiments, dehydrating the first/second phase component and/or ATPS component over different portions allows the target analyte to flow in the first/second direction without the need to flow the sample in the first/second direction. ( See, eg, FIGS. 16D, 16E, and 17 ). In some embodiments, dehydration of the first/second phase component and/or ATPS component over different moieties allows the target analyte to flow more rapidly, leading to faster detection. In some embodiments, dehydration of the first/second phase component and/or ATPS component over different portions allows for increased result reliability. In some embodiments, dehydration of the first/second phase component and/or ATPS component over different portions prevents aggregation of the first/second phase solution component and/or ATPS component ( eg , PEG-salt ATPS). In some embodiments, the first/second phase component and/or the ATPS component are dehydrated in a plurality of segments. In some embodiments, the first/second phase component and/or the ATPS component are dehydrated in a plurality of segments, wherein the first/second phase component and/or the ATPS component comprise a salt solution. In some embodiments, the first/second phase component and/or ATPS component are dehydrated in a plurality of segments, and the first/second phase component and/or ATPS component do not comprise a hydrophobic polymer ( eg , PEG). In some embodiments, dehydrated PEG is not located around the detection region as the PEG-rich phase slows flow within the detection membrane ( see, eg, FIG. 64 ). In some embodiments, the LFA strip comprises a blank spacer near the detection region that does not contain PEG or salt ( see, eg, FIG. 65 ).

In some embodiments, the probe is provided in a probe buffer. In some embodiments, the probe buffer is dehydrated on LFA. In some embodiments, the LFA comprises a probe and a dehydrated probe buffer. In some embodiments, the probe buffer improves the flow of the probe-analyte complex through the LFA.

In some embodiments, dehydration of the ATPS component improves detection limits compared to devices in which the ATPS component is added in liquid form. In some embodiments, the addition of the ATPS component in liquid form dilutes the sample solution from the patient. In some embodiments, dehydration of the ATPS component causes the development of distinct first phase solutions and/or distinct second phase solutions during flow, leading to the target analyte or probe- at the front of the reading fluid reaching the test line and control line. Concentrate the analyte complex to a small volume. In some embodiments, the target analyte and/or probe-analyte complex at the front of the reading fluid reduces the time required for detection.

LFA design/structure

In some embodiments, the LFA strip has a width that does not change from the first end to the second end. In some embodiments, width is defined as the dimension perpendicular to the direction of flow in the plane of length within the LFA. In some embodiments, the first portion of the LFA strip has a first width and the second portion of the LFA strip has a second width, wherein the first width and the second width are different. In some embodiments, the first width is greater than the second width, while in other embodiments the first width is less than the second width. It is contemplated that in certain embodiments, an LFA strip may comprise two or more widths, for example , the strip may narrow continuously or exhibit progressive narrowing at three or more locations. In some embodiments, the first portion comprises a sample pad and the second portion comprises a detection area. In some embodiments, a first portion comprises dehydrated probe buffer and a second portion comprises a detection region ( see, eg, FIG. 62 ). In some embodiments wherein the first portion comprises a dehydrated probe buffer and the second portion comprises a detection region, the detection limit is improved compared to an LFA strip, wherein the width of the portion comprising the sample pad is a width comprising the detection region. It is the same width as the part. In some embodiments, a wider sample pad segment allows more target analyte in the sample to bind to the probe compared to an LFA strip in which the width of the LFA strip does not change. In some embodiments, a wider sample pad segment allows a larger volume of sample, thus more target analyte, to bind to the probe compared to an LFA strip where the width of the LFA strip does not change. ( See, eg, FIG. 63 ).

In some embodiments, the LFA comprises a slope ( eg , the depth of the LFA varies with the length of the LFA). In some embodiments where the LFA does not include a slope, a portion of the probe-analyte complex remains in the sample pad. In some embodiments where the LFA comprises a slope, more probe analyte complexes flow through the LFA than an LFA without a slope ( see, eg, FIG. 66 ).

In some embodiments, the LFA is resorbed at a relatively slow rate to allow the probe to have a large volume to more fully saturate the binding site on the probe, as compared to resolubilizing the probe/probe buffer at a relatively fast rate. Inclusion of moieties comprising lysed dehydrated probes and/or probe buffers leads to improved limits of detection in competition assays. In some embodiments, the dehydrated ATPS or component thereof is disposed downstream from the portion comprising the dehydrated probe and/or probe buffer to provide time for the probe and target analyte to bind to each other before reaching the ATPS or component thereof do. In some embodiments, ATPS (component) concentrates the probe-analyte complex to a smaller volume. In some embodiments, the smaller volume contains more probe-analyte complexes flowing to the test and control lines than in other embodiments, where all of the probe-analyte complexes are distributed over the larger volume of test and The control line is not reached. In some embodiments, the sensitivity and/or reliability of the device is increased by concentrating the probe-analyte complex into fewer reading phases. In some embodiments, detection time is reduced by concentrating the probe-analyte complex into fewer reading phases. See , for example, FIG. 67 , which shows a diagram of one possible design using PEG/salt ATPS.

In some implementations, the LFA is configured according to the structure shown in FIG . 16 . In some implementations, the LFA is configured according to the structure shown in FIG . 20 . In some implementations, the LFA is configured according to the structure shown in Figures 62-70 .

In some embodiments, LFAs are designed for use with probes comprising or complexed with magnetic/paramagnetic particles. In some embodiments, the LFA comprises a paper strip with a fork at the end of the paper strip used to divide the flow of ATPS first and second phase solutions. In some embodiments, the LFA detection region is located at a fork of the fork, and the magnet is located near or at the fork ( see, eg , FIG. 20 ). The magnet concentrates the probe/probe-analyte complex in the fluid flowing into the LFA detection area leading to an increase in diagnostic sensitivity. Conversely, in some embodiments, the probe comprises a magnet or magnetic field and the device comprises magnetic or paramagnetic particles positioned near or at the prong.

In some embodiments, the LFA comprises a 3D structure. In some embodiments, the LFA comprises a layer of a porous matrix that induces a 3D structure. In some embodiments, the 3D structure integrates ATPS with LFA. In some embodiments, ATPS has a long phase separation time ( eg , micellar ATPS), and the phase separation time is improved by using 3D structures ( eg, by increasing the height of the LFA strip). In some embodiments, a mixed phase solution separates into a first phase solution and a second phase solution when the phase solution flows vertically through the LFA ( eg, through a layer of a porous matrix).

In some embodiments, the LFA has a thickness ( eg , a height, depth, or vertical dimension). In some embodiments, the thickness is from about 0.1 mm to about 30 cm. In some embodiments, the thickness is from about 0.1 mm to about 1 mm, from about 0.1 mm to about 10 mm, or from about 0.1 mm to 1 cm. In some embodiments, the thickness is from about 1 mm to about 10 mm, from about 1 mm to about 1 cm, from about 1 mm to about 1.5 cm, from about 1 mm to about 3 cm, from about 1 mm to about 3.5 cm, about 1 mm to about 4 cm, about 1 mm to about 4.5 cm, about 1 mm to about 5 cm, about 1 mm to about 5.5 cm, about 1 mm to about 6 cm, about 1 mm to about 6.5 cm, about 1 mm to about 7 cm, about 1 mm to about 7.5 cm, about 1 mm to about 8 cm, about 1 mm to about 8.5 cm, about 1 mm to about 9 cm, or about 1 mm to about 9.5 cm, or about 1 mm to about It is 10 cm. In some embodiments, the thickness is from about 0.5 cm to about 5 cm.

multipath

In some embodiments, the LFA comprises a first pathway and a second pathway, or at least a first pathway and a second pathway. In some embodiments, the first phase solution preferentially flows through the first pathway and the second phase solution preferentially flows through the second pathway. In some embodiments, the target analyte may not be concentrated in the reading fluid during flow through the paper. In some embodiments, the LFA is designed to redirect flow such that the reading fluid is the first fluid to pass through the detection area at the right time. In some embodiments, redirecting flow comprises introducing a new flow path. In some embodiments, redirecting flow comprises introducing a 3D paper structure. In some embodiments, redirecting flow comprises providing or incorporating a multi-path route in which there is preferential flow along the phase of the fluid. In some embodiments, redirecting flow comprises combining the above methods to redirect flow. In some embodiments, redirecting flow enhances phase separation and/or directs the target analyte to flow through the detection region.

In some embodiments, the LFA is configured to redirect flow by comprising an LFA segment containing a working buffer and an LFA segment containing a dry destination that is introduced after the sample is applied to the LFA. In some embodiments, the LFA is configured to redirect flow by comprising an LFA segment containing a working buffer and an LFA segment containing a dry destination that is introduced after the sample begins to flow through the LFA. In some embodiments, the target is concentrated in the retention fluid, in which case the working buffer reroutes the retention fluid (which makes the reading fluid) to the detection region ( see e.g., Fig. Error! References not found) No. A ). In some embodiments, the working buffer and dry destination segments repel the reading phase containing the concentrated analyte to the detection zone ( FIG. 18B ).

In some embodiments, a plurality of target analytes are present, and the device is configured to partition a first target analyte into a first phase solution and a second target analyte into a second phase solution, wherein the LFA is the first The direction region comprises a first route for the first phase solution, and the LFA comprises a second route for the second phase solution in the second detection region. In some embodiments, the first route consists of a first form of the porous matrix, and the second route consists of a second form of the porous matrix, wherein the first form of the porous matrix and the second form of the porous matrix are different ( eg different porosity, charge, hydrophobicity, three-dimensional structure, etc.). In some embodiments, the first form of the porous matrix is more hydrophobic/hydrophilic than the second form of the porous matrix (see , eg , FIG. 19 ). In some embodiments, the polymer-rich phase solution can preferentially flow through the more hydrophobic stream, while the polymer-poor phase solution containing the concentrated analyte can flow through the more hydrophilic paper. In some embodiments, the target analyte is enriched by “purifying” the polymer-rich phase that may contain contaminants that may affect downstream LFA performance. In some embodiments, one or more of the target analytes of the plurality of target analytes partition into different phases of ATPS.

In some embodiments, the probe comprises magnetic particles and the probe-analyte complex is redirected by one or more magnets. In some embodiments, one or more magnets are placed along the paper strip to concentrate the probe at/to a specific location ( eg , detection area) of the LFA. Or, in some embodiments, the probe comprises a magnet and the probe-analyte complex is redirected by one or more magnetic/paramagnetic particles. In some embodiments, one or more magnetic/paramagnetic particles are placed along a strip of paper to concentrate the probe at/to a specific location ( eg , detection area) of the LFA.

probe

In certain embodiments, a system and/or device described herein comprises and/or a method described herein uses a probe, wherein the probe comprises a binding moiety that binds to a target analyte to form a probe-analyte complex. include As used herein, the terms “target analyte” and “probe-analyte complex” are used interchangeably unless otherwise specified.

In some embodiments, the target analyte alone preferentially partitions into a first phase solution or a second phase solution or an interface of a first phase solution and a second phase solution. In some embodiments, the target analyte alone is extremely partitioned into a first phase solution or a second phase solution or an interface of a first phase solution and a second phase solution.

In some embodiments, the target analyte alone does not preferentially partition into the first phase solution or the second phase solution or the interface of the first phase solution and the second phase solution. In some embodiments, the target analyte alone does not extremely partition into the first phase solution or the second phase solution or the interface of the first phase solution and the second phase solution.

In some embodiments, the probe-analyte complex preferentially partitions into a first phase solution or a second phase solution or an interface of a first phase solution and a second phase solution such that the target analyte (of the probe-analyte complex) is preferentially to be partitioned into the first phase solution or the second phase solution or the interface between the first phase solution and the second phase solution.

In some embodiments, the probe-analyte complex is extremely partitioned into a first phase solution or a second phase solution or an interface of a first phase solution and a second phase solution such that the target analyte (of the probe-analyte complex) is extremely with the first phase solution or the second phase solution or the interface between the first phase solution and the second phase solution.

In some embodiments, the phrase “preferentially cleaved” when used in reference to the cleavage of a target analyte (or probe-analyte complex) into a first/second phase solution of a higher amount of target analyte This indicates that water is placed in the phase solution, which is more preferred than in another phase solution of ATPS.

In some embodiments, the phrase “extremely cleaved” when used in reference to cleavage of a target analyte (or probe-analyte complex) into a first/second phase solution of at least about 90% of the target analyte This indicates that water is placed in the phase solution, which is more preferred than in another phase solution of ATPS.

In some embodiments, a greater amount of the target analyte is partitioned into the first phase solution. In some embodiments, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, More than about 95%, greater than about 98%, and greater than about 99% of the target analyte is partitioned into the first phase solution. In some embodiments, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, More than about 99.9% of the target analyte is resolved into the first phase solution.

In some embodiments, a greater amount of analyte is partitioned into the second phase solution. In some embodiments, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, More than about 95%, greater than about 98%, and greater than about 99% of the target analyte is partitioned into the second phase solution. In some embodiments, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, More than about 99.9% of the target analyte is resolved into the second phase solution.

In some embodiments, a greater amount of analyte partitions at the interface of the first phase solution and the second phase solution. In some embodiments, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, More than about 95%, greater than about 98%, and greater than about 99% of the target analyte are interfacially resolved. In some embodiments, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, More than about 99.9% of the target analyte is interfacially resolved.

In some embodiments, the probe-analyte complex extracts and/or collects application to LFA ( see, eg, FIG. 2 ).

In some embodiments, the probe and/or probe-analyte complex is resuspended in solution ( eg , resuspended solution). In some embodiments, the resuspended solution comprises water. In some embodiments, the resuspended solution comprises saline. In some embodiments, the resuspended solution comprises a buffer. In some embodiments, the resuspended solution comprises a phosphate buffered saline (PBS) solution. In some embodiments, the buffer comprises acetic acid. In some embodiments, the buffer comprises Tris. In some embodiments, the buffer comprises ethylenediaminetetraacetic acid (EDTA). In some embodiments, the buffer comprises boric acid. In some embodiments, the buffer is a borate buffer. In some embodiments, the buffer is a lithium borate buffer. In some embodiments, the buffer is a sodium borate buffer.

In some embodiments, the resuspended solution is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about a pH of 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, or about 12. In some embodiments, the resuspended solution has a pH of about 9.

In some embodiments, a device comprises or is configured to use one probe and/or an assay run on the device uses it. In some embodiments, the device comprises at least 2 different probes or at least 3 different probes or at least 4 different probes or at least 5 different probes or at least 7 different probes or at least 10 different probes or at least 15 different probes or at least Assays that include or are configured to use 20 different probes and/or run on the device use them.

In some embodiments, the probe comprises a material selected from the group consisting of synthetic polymers, metals, minerals, glass, quartz, ceramics, biological polymers, plastics, and combinations thereof. In some embodiments, the probe comprises a polymer selected from the group consisting of polyethylene, polypropylene, nylon (DELRIN®), polytetrafluoroethylene (TEFLON®), dextran, and polyvinyl chloride. In some embodiments, the polyethylene is polyethylene glycol. In some embodiments, the polypropylene is polypropylene glycol. In some embodiments, the probe comprises a biological polymer selected from the group consisting of collagen, cellulose and chitin. In some embodiments, the probe comprises a metal selected from the group consisting of gold, silver, platinum, titanium, stainless steel, aluminum, and alloys thereof. In some embodiments, the probe comprises nanoparticles ( eg , gold nanoparticles, silver nanoparticles, etc. ).

In some embodiments, the probe further comprises a coating. In some embodiments, the coating comprises polyethylene glycol or polypropylene glycol. In some embodiments, the coating comprises polypropylene. In some embodiments, the coating comprises polypropylene glycol. In some embodiments, the coating comprises dextran. In some embodiments, the coating comprises a hydrophilic protein. In some embodiments, the coating comprises serum albumin. In some embodiments, the coating has an affinity for a first phase solution or a second phase solution.

In some embodiments, the amount of target analyte in the sample is very small such that the analyte needs to be substantially concentrated to enable detection by LFA. In certain embodiments, a substantial concentration is achieved at the interface, since the degree of analyte enrichment depends on the volume of the phase in which the analyte is partitioned or concentrated, and the “volume” at the interface is very small compared to the bulk phase.

In some embodiments, the probe preferentially (or extremely) cleaves at the interface to drive the target analyte towards the interface. In some embodiments, probes preferentially (or extremely) cleave at interfaces due to their surface chemistry, wherein the surface chemistry. As a non-limiting example, to drive the probe-analyte complex at the interface of a polymer-salt ATPS system, such as a polyethylene glycol-potassium phosphate (PEG/salt) system, the probe can undergo PEG-PEG interaction with the PEG-rich phase. It is conjugated to (or PEGylated) to PEG to facilitate and/or decorated with a hydrophilic protein to promote hydrophilic interaction with the PEG-deficient phase. Using these optimized probes decorated with specific antibodies or other molecules capable of binding the target, target analytes are captured and collected at the interface. Because the volume of the interface is very small, the analyte is highly concentrated and subjected to subsequent LFA ( FIG. 8 ).

In some embodiments, cleavable gold nanoprobes (GNPs) at the interface of PEG/salt ATPS are prepared, and operating conditions are optimized to enable fast phase separation times with very high recovery of GNPs/analytes. As a non-limiting example, a 100-fold improvement in transferrin (Tf) detection was demonstrated using LFA in combination with interfacial extraction from ATPS (see Example 5).

In some embodiments, the probe-analyte complex is cleaved at the solid-liquid interface in ATPS. In some embodiments, the solid is the wall of the chamber containing ATPS. In some embodiments, the solid is a collector. In some embodiments, the solid comprises a solid polymer. In some embodiments, the solid polymer is selected from polyethylene, cellulose, chitin, nylon, polyoxymethylene (DELRIN®), polytetrafluoroethylene (TEFLON®), polyvinyl chloride, and combinations thereof. In some embodiments, the solid polymer comprises polypropylene ( see, eg, FIG. 9 ). In some embodiments, the probe-analyte complexes are anchored to the solid and are highly concentrated as they exist in small volumes at the solid-liquid interface and are not diluted by the volume of the bulk phase. In some embodiments, the bulk phase is removed without destroying the concentrated analyte and collected by washing with subsequent application to LFA ( FIG. 10 ). In some embodiments, this approach significantly enriches the analyte and enables collection without the use of external forces ( eg , magnets). Alternatively, the probe comprises a magnetic material, and this approach is used with magnets. In some embodiments, such probes are modified to concentrate at the interface for extreme analyte concentrations. As mentioned above, this approach can provide for further separation of the target analyte from other contaminants that are non-specifically enriched by ATPS through the use of magnets. In some embodiments, the ATPS concentration allows the magnetic probe to operate more efficiently because the magnetic probe is first concentrated to a very small volume at a specific location (interface). Thus, smaller magnets or weak magnetic fields are needed to collect concentrated analytes. In some embodiments, the combination of ATPS interfacial concentrations and magnetic probes allows for the development of more effective, faster and less expensive devices compared to current state-of-the-art.

binding moiety

In some embodiments, the binding moiety is a molecule that binds to a target analyte. In some embodiments, the binding moiety is a molecule that specifically binds to a target analyte. In some embodiments, "specifically binds" indicates that a molecule preferentially binds to a target analyte or binds with greater affinity to a target analyte than to other molecules. By way of non-limiting example, an antibody selectively binds to the antigen from which it was generated. Also, by way of non-limiting example, a DNA molecule binds a substantially complementary sequence and not an unrelated sequence under stringent conditions. In some embodiments, “specific binding” can refer to a binding response that is a critical factor in the presence of a target analyte in a heterogeneous population of molecules ( eg, proteins and other biological agents). In some embodiments, the binding moiety binds its particular target analyte and does not bind in significant amounts to other molecules present in the sample.

In some embodiments, the binding moiety is an antibody, lectin, protein, glycoprotein, nucleic acid, monomeric nucleic acid, polymeric nucleic acid, aptamer, aptazyme, small molecule, polymer, lectin, carbohydrate, polysaccharide, sugar, lipid and any thereof is selected from the group consisting of a combination of In some embodiments, a binding moiety is a molecule capable of pairwise binding a target analyte.

In some embodiments, the binding moiety is an antibody or antibody fragment. Antibody fragments include Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, Fv', Fd, Fd', scFv, hsFv fragments, cameloid antibodies, diabodies, and other fragments described below, but , but not limited thereto.

In some embodiments, "antibody" refers to a protein consisting of one or more polypeptides encoded by an immunoglobulin gene or fragment of an immunoglobulin gene. As used herein, the terms “antibody” and “immunoglobulin” are used interchangeably unless otherwise specified. In some embodiments, the immunoglobulin gene is an immunoglobulin constant region gene. In some embodiments, the immunoglobulin gene is a kappa, lambda, alpha, gamma, delta, epsilon or mu constant region gene, including but not limited to. In some embodiments, the immunoglobulin gene is an immunoglobulin variable region gene. In some embodiments, the immunoglobulin gene comprises a light chain. In some embodiments, the light chain is selected from a kappa light chain, a lambda light chain, or a combination thereof. In some embodiments, the immunoglobulin gene comprises a heavy chain. In some embodiments, light chains are classified as gamma, mu, alpha, delta or epsilon, which also correspond to the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

In some embodiments, the immunoglobulin comprises a tetramer. In some embodiments, a tetramer consists of two identical pairs of polypeptide chains, each pair comprising one “light” chain (about 25 kDa) ?? It has one "heavy" chain (about 50-70 kDa). In some embodiments, the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light (V _L ) and variable heavy (V _H ) chain refer to these light and heavy chains, respectively.

In some embodiments, the antibody comprises pure immunoglobulin. In some embodiments, the antibody is selected from a number of well characterized fragments produced by digestion with various peptidases. In some embodiments, the peptidase is pepsin. In some embodiments, pepsin digests a disulfide bond at the hinge region to produce a dimer of Fab, F(ab)' ₂ , which is itself a light chain bound to V _H -C _H 1 by a disulfide bond. In some embodiments, F(ab)′ ₂ is reduced under gentle conditions to break the disulfide bond in the hinge region to convert the (Fab′) ₂ dimer to a Fab′ monomer. In some embodiments, the Fab' monomer consists essentially of a Fab having a portion of the hinge region. In some embodiments, Fab' fragments are synthesized de novo chemically or by using recombinant DNA methodologies. In some embodiments, antibody fragments are produced by modification of whole antibodies. In some embodiments, antibody fragments are synthesized de novo using recombinant DNA methodology. In some embodiments, the antibody comprises a single chain antibody (an antibody that exists as a single polypeptide chain). In some embodiments, the antibody comprises a single chain Fv antibody (sFv or scFv) in which the variable heavy and variable light chains are joined together (either directly or via a peptide linker) to form a continuous polypeptide. In some embodiments, the antibody comprises a single chain Fv antibody. In some embodiments, an antibody comprises a covalently linked V _H -V _L heterodimer that can be expressed from a nucleic acid comprising V _H - and V _L -coding sequences that are directly attached or joined by a peptide-encoding linker. In some embodiments, V _H and V _L are linked to each as a single polypeptide chain, and the V _H and V _L domains are non-covalently linked. In some embodiments, the Fab appears on a phage, wherein one of the chains (heavy or light chain) is fused to a g3 capsid protein and the complementary chain is exported to the periplasm as a soluble molecule. In some embodiments, both chains may be encoded on the same or different replicons. In some embodiments, two antibody chains of each Fab molecule are post-translationally assembled and the dimer is introduced into the phage particle, eg, via binding of one of the chains to g3p. In some embodiments, the antibody is displayed on phage.

In some embodiments, the binding moiety comprises an aptamer. In some embodiments, an aptamer comprises an antibody-analog formed from a nucleic acid. In some embodiments, the aptamer does not require binding of a label detected in some assays, eg, a nano-CHEM-FET in which the reconstitution is directly detected. In some embodiments, the binding moiety comprises an aptazyme. In some embodiments, an aptazyme comprises an enzyme analog formed from a nucleic acid. In some embodiments, the aptazyme functions to alter the composition to capture a specific molecule only in the presence of a second specific analyte.

The term “small molecule” or “small organic molecule” refers to a molecule of a size comparable to that of an organic molecule generally used in pharmaceuticals. This term excludes biological macromolecules ( eg proteins, nucleic acids, etc. ). Preferred small molecules vary in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In some embodiments, antibody fragments are derived via proteolytic digestion of a pure antibody. In some embodiments, antibody fragments are produced directly by recombinant host cells. In some embodiments, the Fab, Fv or ScFv antibody fragment is internally expressed and is expressed in E. It is secreted from E. coli to allow for the easy production of these large quantities. In some embodiments, antibody fragments are isolated from antibody phage libraries. In some embodiments, the Fab'-SH fragment is E. coli and can be chemically coupled to form F(ab′) ₂ fragments. In some embodiments, the F(ab′) ₂ fragment is isolated directly from recombinant host cell culture. In some embodiments, Fab and F(ab′) ₂ fragments increase half-life in vivo . In some embodiments, the Fab and F(ab') ₂ fragments comprise a salvage receptor binding epitope residue. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody of choice is a single chain Fv fragment. In some embodiments, the Fv or sFv has a pure binding site free of constant regions; Therefore, they are suitable for reduced non-specific binding during in vivo use. In some embodiments, an antibody fragment is a “linear antibody”. In some embodiments, a linear antibody fragment is monospecific. In some embodiments, the linear antibody fragment is bispecific.

In some embodiments, the antibody fragment is a diabody. In some embodiments, a diabody is an antibody fragment having two antigen binding sites, which may be bivalent or bispecific.

In some embodiments, the antibody fragment is a single-domain antibody. In some embodiments, a single-domain antibody is an antibody fragment comprising part or all of the heavy chain variable domain or part or all of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody.

In some embodiments, the probe comprises a detectable label. In some embodiments, the probe has detectable properties. Detectable labels/detectable properties include any composition by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical processing. Exemplary useful labels include, but are not limited to: fluorescent nanoparticles ( eg, quantum dots (Qdots)) , fluorescent dyes ( eg , fluorescein, Texas red, rhodamine, green fluorescent protein, etc., see: e.g., Molecular Probes, Eugene, Oregon, USA), radiolabeled ( e.g. , ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³² P, ⁹⁹ Tc, ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ¹¹¹ In, ^113m In, ⁹⁷ Ru, ⁶² Cu, 64lCu, ⁵² Fe, ^52m Mn, ⁵¹ Cr, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁷⁷ As, ⁹⁰ Y, ⁶⁷ Cu, ¹⁶⁹ Er, ¹²¹ Sn, ¹²⁷ Te, ¹⁴² Pr, ¹⁴³ Pr, ¹⁹⁸ Au, ¹⁹⁹ Au, ¹⁶¹ Tb, ¹⁰⁹ Pd, ¹⁶⁵ Dy, ¹⁴⁹ Pm, ¹⁵¹ Pm, ¹⁵³ Sm, ¹⁵⁷ Gd, ¹⁵⁹ Gd, ¹⁶⁶ Ho, ¹⁷² Tm, ¹⁶⁹ Yb, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁰⁵ Rh, ¹¹¹ Ag, etc.), enzymes ( eg horseradish peroxidase, alkaline phosphatase and others commonly used in ELISA), various colorimetric labels, magnetic or paramagnetic labels ( eg magnetic and/or paramagnetic nanoparticles), spin labels, radio-opaque labels, etc.

Alternatively or additionally, the probe binds to another particle comprising a detectable label. Alternatively or additionally, the probe binds to another particle with detectable properties. In some embodiments, the probe provides a detectable signal in a detection region ( eg , a test line, a control line). In some embodiments, the detectable label/property is selected from the group consisting of a colorimetric label/property, a fluorescent label/property, an enzymatic label/property, a colorgenic label/property, and a radioactive label/property. In some embodiments, the probe is a gold nanoparticle and the detectable property is color. In some embodiments, the color is selected from orange, red and purple.

In some embodiments, the probe comprises magnetic and/or box-shaped particles. In some embodiments, the magnetic particles comprise a material selected from the group consisting of iron, nickel, cobalt, and combinations thereof. In some embodiments, the magnetic particles comprise an alnico, aluminum-nickel-cobalt alloy. In some embodiments, the magnetic particles comprise an alloy of a rare earth metal. In some embodiments, the rare earth metal is selected from scandium, yttrium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, or combinations thereof. In some embodiments, the magnetic component comprises a rare earth mineral. In some embodiments, the rare earth mineral comprises magnetite. In some embodiments, the magnetic particles comprise iron. In some embodiments, the magnetic particle comprises a ferromagnetic material. In some embodiments, the ferromagnetic material is a soft ferromagnetic material. In some embodiments, the soft ferromagnetic material comprises annealed iron. In some embodiments, the soft ferromagnetic material comprises zinc, nickel, manganese, or combinations thereof. In some embodiments, the soft ferromagnetic material is selected from manganese-zinc ferrite (MnaZn(1-a)Fe ₂ O ₄ ) and nickel-zinc ferrite (NiaZn(1-a)Fe ₂ O ₄ ). In some embodiments, the ferromagnetic material is a hard ferromagnetic material. In some embodiments, the hard ferromagnetic material comprises alnico or ferrite. In some embodiments, the hard ferromagnetic material is strontium ferrite, SrFe ₁₂ O ₁₉ (SrO·6Fe ₂ O ₃ ), barium ferrite, BaFe ₁₂ O ₁₉ (BaO·6Fe ₂ O ₃ ) and cobalt ferrite, CoFe ₂ O ₄ (CoO) ㆍFe ₂ O ₃ ). In some embodiments, the magnetic component comprises ferrite. In some embodiments, the ferrite is selected from hematite (Fe ₂ O ₃ ). In some embodiments, the magnetic component comprises ferrite. In some embodiments, the ferrite is selected from magnetite (Fe ₃ O ₄ ). In some embodiments, the magnetic particles comprise Fe ₃ O ₄ . In some embodiments, the magnetic particles comprise Fe ₂ O ₃ . In some embodiments, the magnetic particles consist essentially of Fe3O4. In some embodiments, the magnetic particles consist essentially of Fe ₂ O ₃ .

In some embodiments, the magnetic particles have a shape selected from, but not limited to, a sphere, a cube, an ellipse, a rod, a capsule shape, a tablet shape, and a static random shape.

In some embodiments, the magnetic particles comprise an average diameter of from about 1 nm to about 100 μm. In some embodiments, the magnetic particles comprise an average diameter of about 20 nm to about 1 μm. In some embodiments, the magnetic particles comprise an average diameter of about 10 nm to about 50 nm. In some embodiments, the magnetic particles comprise an average diameter of from about 100 nm to about 750 nm. In some embodiments, the magnetic particles comprise an average diameter of about 500 nm. In some embodiments, the magnetic particles comprise an average diameter of from about 1 μm to about 15 μm. In some embodiments, the magnetic particles comprise an average diameter of about 2 μm to about 10 μm. In some embodiments, the magnetic particles comprise an average diameter of from about 1 μm to about 5 μm. In some embodiments, the magnetic particles comprise an average diameter of about 1 μm. In some embodiments, the magnetic particle comprises an average diameter of from about 0.5 μm to about 20 μm at its widest point.

21 depicts one illustrative, but non-limiting example of how to use magnetic probes to enrich analytes in ATPS. Using an antibody (or other binding moiety) specific for the target analyte, the target is captured and concentrated with a magnetic probe in one of the bulk phases in ATPS. In complex samples containing different types of molecules, ATPS can non-specifically enrich for molecules with physicochemical properties similar to the target analyte or probe. However, through the use of magnets or external magnetic fields, further enrichment and isolation of the target analyte bound to the magnetic capture probe is achieved prior to detection.

In some embodiments, the device includes a magnetic material. In some embodiments, the magnetic material is a source of a magnetic field. In some embodiments, the magnet comprises a material selected from magnetite, magnetite, cobalt, nickel, manganese, aluminum, gadorium, dysprosium, ceramic ( eg , ferrite), and alnico. In some embodiments, the magnet comprises ore. In some embodiments, the magnet comprises iron ore. In some embodiments, the magnet comprises a rare earth magnet. In some embodiments, the rare earth magnet is selected from a samarium-cobalt magnet and a neodymium-iron-boron magnet (NIB). In some embodiments, the magnet has a magnetic field strength of about 0.1 gauss, about 0.2 gauss, about 0.3 gauss, about 0.4 gauss, about 0.5 gauss, about 0.6 gauss, about 0.7 gauss, about 0.8 gauss, about 0.9 gauss, or about 1 gauss. include In some embodiments, the magnet has a magnetic field strength of about 1 Gauss, about 2 Gauss, about 3 Gauss, about 4 Gauss, about 5 Gauss, about 6 Gauss, about 7 Gauss, about 8 Gauss, about 9 Gauss, or about 10 Gauss. In some embodiments, the magnet is about 10 gauss, about 20 gauss, about 30 gauss, about 40 gauss, about 50 gauss, about 60 gauss, about 70 gauss, about 80 gauss, about 90 gauss, about 100 gauss, about 110 gauss, about 120 gauss, about 130 gauss, about 140 gauss, about 150 gauss, about 160 gauss, about 170 gauss, about 180 gauss, about 190 gauss, about 200 gauss, about 250 gauss, about 300 gauss, about 350 magnetic field strengths of gauss, about 400 gauss, about 450 gauss, or about 500 gauss. In some embodiments, the magnet comprises a magnetic field strength of about 100 Gauss.

In some embodiments, the magnet is configured to promote and/or increase partitioning of the target analyte into a first phase solution or a second phase solution. In some embodiments, the magnet is configured to facilitate and/or increase flow of a target analyte through the LFA. In some embodiments, the magnet is attachable to and/or detachable from the device. In some embodiments, magnets are provided in/on the collector.

In some embodiments, the device comprises at least one target analyte or at least 2 different target analytes or at least 3 different target analytes or at least 4 different target analytes or at least 5 different target analytes or at least 7 different target analytes and/or is configured to use and/or include one or more probes that interact with a target analyte or at least 10 different target analytes or at least 15 different target analytes or at least 20 different target analytes.

Target analyte/sample

In various embodiments, the devices, systems and/or methods described herein detect and/or quantify one or more target analyte(s). In some embodiments, the target analyte is selected from a protein, an antigen, a biomolecule, a sugar moiety, a lipid, a nucleic acid, a sterol, a small organic molecule, and combinations thereof. In some embodiments, the target analyte is from an organism selected from the group consisting of plants, animals, viruses, fungi, protozoa, and bacteria.

In some embodiments, the target analyte comprises a biological molecule. In some embodiments, the biological molecule is selected from the group consisting of nucleic acids, proteins, lipids, small molecules, metabolites, sugars, antibodies, antigens, enzymes, and combinations thereof. In some embodiments, the sugar is lactose.

The terms “protein”, “peptide” and “polypeptide” are used interchangeably herein. In some embodiments, protein refers to a polymer of amino acid residues. In some embodiments, the protein is a naturally occurring amino acid polymer as well as an amino acid polymer wherein one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid.

The terms “nucleic acid” or “oligonucleotide” are used interchangeably herein. In some embodiments, a nucleic acid comprises at least two nucleotides covalently linked together. In some embodiments, the nucleic acids of the invention are single-stranded. In some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid is triple stranded. In some embodiments, the nucleic acid comprises a phosphodiester linkage. In some embodiments, a nucleic acid comprises a nucleic acid analog. In some embodiments, nucleic acid analogs contain linkages other than phosphodiester linkages and/or backbones in addition to phosphodiester linkages, such as, but not limited to, phosphoramide, phosphorothioate, phosphorodithio ate or O-methylphosphoroamidite. In some embodiments, the nucleic acid analog is selected from a nucleic acid analog having a backbone selected from a positive backbone, a nonionic backbone, and a non-ribose backbone. In some embodiments, the nucleic acid contains one or more carbocyclic sugars. In some embodiments, the nucleic acid comprises a modification of its ribose-phosphate backbone. In some embodiments, such modifications are performed to facilitate the addition of additional moieties such as labels. In some embodiments, such modifications are performed to increase the stability and half-life of these molecules in a physiological environment.

In some embodiments, the biological molecule is derived from a virus. In some embodiments, the biological molecule is a viral particle. In some embodiments, the viral particle is derived from a virus, wherein the virus is selected from the group consisting of influenza virus, immunodeficiency virus, respiratory virus. In some embodiments, the virus is adenovirus, herpes virus, papillomavirus, varicella virus, parvovirus, astrovirus, calicivirus, picornavirus, flavivirus, togavirus, hepevirus, retrovirus, orthomyxoviruses, arenaviruses, bubunviruses, filoviruses, paramyxoviruses, rhabdoviruses and reoviruses. In some embodiments, the virus is selected from rotavirus, orbivirus, cortivirus, Marburg virus, rubella virus, norwalk virus, rhinovirus, Epstein Barr virus and cytomegalovirus. In some embodiments, the virus is smallpox virus. In some embodiments, the virus is a hepatitis virus. In some embodiments, the virus is a rabies virus. In some embodiments, the virus is a respiratory syncytial virus. In some embodiments, the virus is a mumps virus. In some embodiments, the virus is a measles virus. In some embodiments, the virus is Ebola virus. In some embodiments, the virus is a poliovirus. In some embodiments, the virus is selected from seasonal H1N1, porcine H1N1, H3N2 influenza. In some embodiments, the virus is a bacteriophage. In some embodiments, the bacteriophage is an M13 bacteriophage.

In some embodiments, the biological molecule is from a bacterium. In some embodiments, the biological molecule is a bacterial particle. In some embodiments, the bacterium is from a genus selected from Streptococcus, Chlamydia , Mycobacterium, and Neisseria . In some embodiments, the bacterium is Streptococcus mutans (Streptococcus mutans) . In some embodiments , the bacteria are Bacillus , Bordetella , Borrelia , Brucella , Campylobacter , Chlamydophila , Clostridium ) , Corynebacterium , Enterococcus , Escherichia , Francisella , Haemophilus , Helicobacter , Legionella , Leptospira , Listeria , Mycobacterium, Mycoplasma , Neisseria, Pseudomonas , Rickettsia , Salmonella , Shigella , Staphylococcus Staphylococcus , Treponema, Vibrio and It is of a genus selected from the group consisting of Yersinia . In some embodiments, the bacterium is Streptococcus Pneumoniae (Streptococcus pneumoiae ) is. In some embodiments, the bacterium is Chlamydia trachomatis . In some embodiments, the bacterium is Neisseria Gonorrhoeae (Neisseria gonorrhoeae ). In some embodiments, the bacterium is Mycobacterium tuberculosis .

In some embodiments, the bacterium is Staphylococcus aureus. In some embodiments, the bacterium is Staphylococcus saprophyticus. In some embodiments, the bacterium is Escherichia coli. In some embodiments, the bacterium is Bacillus anthracis. In some embodiments, the bacterium is Clostridium botulinum. In some embodiments, the bacterium is Helicobacter pylori.

In some embodiments, the bacteria are oral bacteria. In some embodiments, oral bacteria cause periodontal disease. In some embodiments, the oral bacteria are Porphyromonas gingivalis , Bacteroides Forsythus ( Bacteroides forsythus ), Treponema denticola ( Treponema ) denticola ), Prevotella intermedia ) , Fusobacterium Fusobacterium nucleatum , Microaerophile bacteria Actinomyces actinomycetemcomitans , Campylobacter _ _ Lectus ( Campylobacter rectus ) , Eikenella corrodens and Eubacterium species . _

In some embodiments, the sample or target analyte is from a protozoan. In some embodiments, the protozoa is suffering from abemaic infection, trichomoniasis, toxoplasmosis, cryptosporidiosis, trichomoniasis, Shajas disease, leishmaniasis, sleep sickness, Avema dysentery, Avema keratitis, and basic amoeba meningoencephalitis. cause a disease selected from the group consisting of In some embodiments, the protozoa is from the genus Plasmodium, which is known to cause malaria. In some embodiments, the target analyte is Plasmodium lactate dehydrogenase (pLDH).

In some embodiments, the target analyte is a food allergen. In some embodiments, the food allergen is a plant biomolecule. In some embodiments, the plant biomolecule is a plant protein. In some embodiments, the plant protein is gluten. In some embodiments, the food allergen is a plant biomolecule. In some embodiments, the plant biomolecule is a plant protein. In some embodiments, the plant protein is gluten. In some embodiments, the food allergen is an animal biomolecule. In some embodiments, the animal biomolecule is a polysaccharide. In some embodiments, the polysaccharide is lactose.

In some embodiments, the target analyte is a protein. In some embodiments, the protein is present in and/or derived from a biological sample ( eg , blood, plasma, serum, urine, saliva, tears/eye drops, bodily fluid collected with a swab). In some embodiments, the protein is a biomarker. In some embodiments, the protein is a biomarker of a disease or condition. In some embodiments, the target analyte is troponin or a fragment thereof. In some embodiments, the target analyte is transferrin or a fragment thereof.

In some embodiments, the device is configured to detect/quantify a plurality of analytes in a sample. In some embodiments, ATPS enriches a plurality of analytes with similar physicochemical properties. In some embodiments, LFAs are capable of testing multiple target analytes. In some embodiments, the LFA comprises a plurality of test lines. In some embodiments, the LFA comprises a first test line having a first capture moiety specific for a first target analyte and a second test line having a second capture moiety specific for a first target analyte, wherein the second The first capture moiety and the second capture moiety are different. The apparatus comprises about 1 test line, about 2 test lines, about 3 test lines, about 4 test lines, about 5 test lines, about 6 test lines, about 7 test lines, about 8 test lines, It may include about 9 test lines or about 10 test lines.

In some embodiments, the target analyte is present in a sample. In some embodiments, the target analyte is derived from a sample. In some embodiments, a target analyte is isolated from a sample. In some embodiments, a target analyte is purified from a sample.

In some embodiments, the sample is selected from the group consisting of: tissue/fluid from a biological organism; food samples; chemical samples; drug samples; environmental samples; and combinations thereof.

In some embodiments, the food sample is selected from nuts, dairy products, wheat products, soy products, and egg products. In some embodiments, the food sample is soy. In some embodiments, the food product is a fruit.

In some embodiments, the sample is selected from the group consisting of: a blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample, a tear/ocular fluid sample, a swab sample, and combinations thereof.

In some embodiments, the sample is derived from a source selected from the group consisting of bacteria, viruses, protozoa, algae, fungi, drugs, pathogens, toxins, environmental pollutants and components thereof, and combinations thereof. In some embodiments, the sample is a bacterial warfare agent. In some embodiments, the sample is a water sample. In some embodiments, the water sample is from a water testing facility. In some embodiments, the water sample is from a water purification facility.

In some embodiments, the sample is derived from a building, an inanimate structure, or a chemical solution. In some embodiments, the sample is derived from carpets, paints, preservative wood, deodorants, cleaning products, cosmetics, apparel, insect repellents, air fresheners, floors, walls, ceilings, linoleum, plastics, konjac sprays and dental gels/pests. .

II. Way

Disclosed herein are methods of detecting one or more target analyte(s) in a sample. Also provided is a method of quantifying one or more target analyte(s) in a sample comprising applying the sample to any one of the devices disclosed herein and quantifying the target analyte(s) in a lateral flow assay. do. Methods for enriching a target analyte in a sample are also provided. In various embodiments, a method comprises applying a sample to any one of the devices disclosed herein and detecting the presence or absence of a target analyte in a lateral flow assay. In various embodiments, a method comprises applying a sample to any one of the devices disclosed herein; and concentrating the target analyte(s) into an aqueous two-phase system and/or in a lateral flow assay. Disclosed herein are methods of cleaving/isolating one or more target analyte(s) in a sample. In various embodiments, a method comprises applying a sample to any one of the devices disclosed herein; and partitioning/separating the target analyte into an aqueous two-phase system and/or in a lateral flow assay. In some embodiments, the method(s) comprises subjecting the sample to ATPS. In some embodiments, the method(s) comprises subjecting the sample to LFA. In some embodiments, the method comprises subjecting the sample to LFA, wherein the LFA and ATPS are integrated.

In some embodiments, the method comprises subjecting the sample to ATPS. In some embodiments, the method comprises subjecting the sample to LFA, wherein the ATPS is dehydrated on the LFA.

In some embodiments, the target analyte is concentrated in a phase solution or interface of ATPS. In some embodiments, the method comprises extracting the concentrated target analyte from the phase solution and subjecting it to LFA. In some embodiments, a method comprises contacting ATPS with a collector, eg , as described herein, wherein the target analyte, probe, and/or probe-analyte complex has affinity for the collector , interact with the collector. In some embodiments, the method comprises detecting the target analyte on a collector when it is inserted into the detection well ( FIG. 11 ).

In some embodiments, the method further comprises extracting a target analyte from the sample. In some embodiments, extracting the target analyte may comprise the use of ATPS. In some embodiments, extracting the target analyte can include disrupting, dissolving, milling, blending, mincing, stirring, centrifuging, or cleaving the sample. In some embodiments, to test for food allergens from solid food, a sample is first ground. In some embodiments, the target analyte is extracted from the milled sample using an extraction buffer. In some embodiments, the milled sample is mixed with an ATPS solution to extract a target analyte from the sample.

In some embodiments, the method further comprises mixing the sample and/or target analyte with the probe. In some embodiments, the method further comprises mixing the sample or target analyte with the probe to generate a probe-analyte complex prior to subjecting the sample and/or target analyte to ATPS and/or LFA. In some embodiments, the method further comprises washing the probe-analyte complex prior to subjecting the sample and/or target analyte to ATPS and/or LFA.

purpose

In some aspects, the methods and devices described herein are used to detect a target analyte in a sample. In some aspects, the methods and devices described herein are used to quantify a target analyte in a sample. In some aspects, the methods and devices described herein are used to detect and quantify a target analyte in a sample.

In some aspects, the methods described herein include diagnosing the condition or disease. In some embodiments, it can include observing a result for LFA of a device disclosed herein and determining a diagnosis based on the result. In certain instances, assay results may be critical for diagnosis, while in other contexts, assay results may be evaluated in the context of differential diagnosis. In some embodiments, diagnosing the condition or disease requires additional testing using additional devices.

infectious disease

In some embodiments, the condition or disease is an infectious disease. In some embodiments, the infectious disease is a respiratory infection. In some embodiments, the infectious disease is influenza. In some embodiments, the infectious disease is a tropical disease.

In some embodiments, the infectious disease is selected from the common cold, cervical cancer, herpes, smallpox, chickenpox, herpes zoster, hepatitis, rabies, mumps and polio. In some embodiments, the infectious disease is tuberculosis. In some embodiments, the infectious disease is Ebola. In some embodiments, the infectious disease is malaria. In some embodiments, the infectious disease is measles. In some embodiments, the infectious disease is pertussis. In some embodiments, the infectious disease is tetanus. In some embodiments, the infectious disease is meningitis. In some embodiments, the infectious disease is syphilis. In some embodiments, the infectious disease is hepatitis B infection.

sexually transmitted infections

In some embodiments, the condition or disease is a sexually transmitted infection. In some embodiments, the sexually transmitted infection is chlamydia, gonorrhea, herpes (HSV-1, HSV-2), human papillomavirus infection, syphilis, hepatitis B, hepatitis C, hepatitis A, bacterial vaginosis, crab, scabies, trichomoniasis. ichthyosis, amoebosis, cryptosporidiosis, rambler trichomoniasis, candidiasis and bacterial dysentery.

periodontal disease

In some embodiments, the condition or disease is periodontal disease. In some embodiments, the periodontal disease is gingivitis. In some embodiments, the periodontal disease is periodontitis. In some embodiments, the periodontal disease is caused by the presence of the bacterium Streptococcus mutans in the oral cavity.

Allergen/Toxin

In some embodiments, the target analyte is a contaminant in a liquid or food sample. In some embodiments, the target analyte is an allergen in a liquid or food sample. In some embodiments, the allergen is selected from dairy allergy, egg allergy, soy allergy, wheat allergy, shellfish allergy and nut allergy. In some embodiments, the nut allergy is a peanut allergy. In some embodiments, the condition or disease is an intolerance or sensitivity to a food or food ingredient. In some embodiments, the condition or disease is gluten intolerance.

In some embodiments, the target analyte is an environmental toxin. Environmental toxins are selected from pesticides and herbicides. In some embodiments, the environmental toxin is selected from molds and fungi. In some embodiments, the environmental toxin is selected from polychlorinated biphenyls (PCBs) and phthalates. In some embodiments, the environmental toxin is selected from volatile organic compounds, dioxins, asbestos, heavy metals, chloroform and chlorine.

Various embodiments contemplated herein may include, but are not limited to, one or more of the following and combinations thereof:

Embodiment 1: A device for detecting and/or quantifying a target analyte in a sample, the device comprising: a: a lateral flow assay (LFA); and b: an aqueous two-phase system (ATPS), wherein the ATPS comprises a mixed phase solution separated into a first phase solution and a second phase solution.

Embodiment 2: The apparatus of embodiment 1, wherein the separation of the mixed phase into a first phase solution and a second phase solution occurs in an LFA.

Embodiment 3: The apparatus of Embodiment 1, wherein the separation of the mixed phase into a first phase solution and a second phase solution does not occur in the LFA.

Embodiment 4: The device of any of Embodiments 1-3, wherein the target analyte is contacted with a mixed phase solution, and the target analyte is partitioned into a first phase solution or a second phase solution.

Embodiment 5: The device of any of Embodiments 1-3, wherein the target analyte is contacted with a mixed phase solution, and the target analyte is partitioned at the interface of the first phase solution and the second phase solution.

Embodiment 6: The device of embodiment 4 or 5, wherein the target analyte is enriched upon cleavage.

Embodiment 7: The device of any of Embodiments 1-6, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a polymer.

Embodiment 8: The device of any of Embodiments 1-6, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a salt.

Embodiment 9: The device of Embodiments 7 or 8, wherein the micellar solution comprises a surfactant.

Embodiment 10: The device of embodiment 9, wherein the surfactant is selected from the group consisting of ionic surfactants, surfactants comprising zwitter ions, and nonionic surfactants.

Embodiment 11: The device of any of Embodiments 7-10, wherein the micellar solution comprises Triton-X.

Embodiment 12: The device of any of Embodiments 1-6, wherein the first phase solution comprises a first polymer and the second phase solution comprises a second polymer.

Embodiment 13: The device of embodiment 12, wherein the first/second polymer is selected from polyethylene glycol, polypropylene glycol and dextran.

Embodiment 14: The device of any of Embodiments 1-6, wherein the first phase solution comprises a polymer and the second phase solution comprises a salt.

Embodiment 15: The device of embodiment 14, wherein the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate.

Embodiment 16: The device of any of Embodiments 1-6, wherein the first phase solution is selected from component 1 of Table 1 and the second phase solution is selected from component 2 of Table 1.

Embodiment 17: The method of any one of embodiments 1 to 16, wherein the target analyte is selected from the group consisting of proteins, antigens, biomolecules, small organic molecules, sugar moieties, lipids, nucleic acids, sterols and combinations thereof. Device.

Embodiment 18: The device of embodiment 17, wherein the target analyte is from an organism selected from the group consisting of plants, animals, viruses, protozoa, fungi and bacteria.

Embodiment 19: The device of any of embodiments 1-17, wherein the device further comprises a probe, wherein the probe interacts with the target analyte.

Embodiment 20: The device according to embodiment 19, wherein the device comprises at least one target analyte or at least two different target analytes or at least 3 different target analytes or at least 4 different target analytes or at least 5 different target analytes A device comprising one or more probes that interact with water or at least 7 different target analytes or at least 10 different target analytes or at least 15 different target analytes or at least 20 different target analytes.

Embodiment 21: The device according to any of embodiments 19 or 20, wherein the device comprises at least 2 different probes or at least 3 different probes or at least 4 different probes or at least 5 different probes or at least 7 different probes or at least 10 different probes. or at least 15 different probes or at least 20 different probes.

Embodiment 22: The device of embodiment 19, wherein the probe is selected from the group consisting of synthetic polymers, metals, minerals, glass, quartz, ceramics, biological polymers, plastics, and combinations thereof.

Embodiment 23: The probe of embodiment 19, wherein the probe comprises a polymer selected from the group consisting of polyethylene, polypropylene, cellulose, chitin, nylon, polyoxymethylene, polytetrafluoroethylene, polyvinyl chloride, and combinations thereof. to do, device.

Embodiment 24: The device of embodiment 19, wherein the probe comprises a biological polymer selected from the group consisting of dextran, polypropylene, polyethylene glycol, and combinations thereof.

Statement 25: The device of statement 19, wherein the probe comprises a metal selected from the group consisting of gold, silver, platinum, and combinations thereof.

Embodiment 26: The device of embodiment 19, wherein the probe comprises a nanoparticle.

Embodiment 27: The device of embodiment 19, wherein the nanoparticles are gold nanoparticles.

Embodiment 28: The device of any of embodiments 17-27, wherein the probe comprises a coating.

Embodiment 29: The device of embodiment 28, wherein the coating comprises a polymer selected from polypropylene glycol and polyethylene glycol.

Embodiment 30: The device of embodiment 28, wherein the coating comprises dextran.

Embodiment 31: The device of embodiment 28, wherein the coating comprises a hydrophilic protein.

Embodiment 32: The device of embodiment 28, wherein the coating comprises serum albumin.

Embodiment 33: The device of any of embodiments 28-32, wherein the coating has an affinity for the first phase solution or the second phase solution.

Embodiment 34: The device of any one of embodiments 28-33, wherein the probe further comprises a binding moiety that binds to a target analyte.

Embodiment 35: The device of embodiment 34, wherein the binding moiety is selected from the group consisting of an antibody, a lectin, a protein, a glycoprotein, a nucleic acid, a small molecule, a polymer, a lipid, and combinations thereof.

Embodiment 36: The device of embodiment 34, wherein the binding moiety is an antibody or antibody fragment.

Embodiment 37: The device of any of embodiments 18-35, wherein the probe comprises a magnetic particle.

Embodiment 38: The device of embodiment 37, further comprising a magnet.

Embodiment 39: The device of embodiment 38, wherein the magnet is configured to promote and/or increase partitioning of the target analyte into a first phase solution or a second phase solution.

Embodiment 40: The device of embodiment 38, wherein the magnet is configured to facilitate and/or increase flow of a target analyte through the LFA.

Statement 41: The device of any of statements 38-40, wherein the magnet is attachable and/or detachable to the device.

Embodiment 42: The device of any one of embodiments 1-41, further comprising a collector configured to be placed in contact with ATPS, wherein the target analyte is disposed with the collector and the first phase solution and/or the second phase solution The device, which is divided at the interface of

Statement 43: The device of statement 42, wherein the collector comprises a material selected from plastic, mesoporous material, silica, polypropylene, magnet, porous material, grooved material, and combinations thereof.

Embodiment 44: The device of any of embodiments 19-43, wherein the probe comprises a detectable label.

Embodiment 45: The device of embodiment 44, wherein the detectable label is selected from the group consisting of a colorimetric label, a fluorescent label, an enzymatic label, a colorigenic label, a radioactive label, and combinations thereof.

Embodiment 46: The device of any of embodiments 1-45, wherein the LFA comprises a porous matrix.

Embodiment 47: The device of embodiment 46, wherein the porous matrix is sufficiently porous to allow the mixed phase solution, the first phase solution, the second phase solution, and/or the target analyte to flow through the LFA.

Embodiment 48: The method of embodiment 46 or 47, wherein the porous matrix is sufficient to allow the mixed phase solution, the first phase solution, the second phase solution and/or the target analyte to flow vertically and/or horizontally through the LFA. length and/or depth.

Embodiment 49: The method of any one of Embodiments 46-48, wherein the first phase solution flows through the porous matrix at a first rate, and the second phase solution flows through the porous matrix at a second rate, and wherein wherein the first speed and the second speed are different.

Embodiment 50: The material of any of Embodiments 46-49, wherein the porous matrix is selected from cellulose, glass fibers, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and combinations thereof. A device comprising a.

Embodiment 51: The device of any one of embodiments 1-50, wherein the LFA comprises a target analyte capture moiety, wherein the target analyte capture moiety interacts with the target analyte.

Embodiment 52: The device of any of embodiments 1-51, configured to provide a competition assay when the LFA is used.

Embodiment 53: The device of any one of embodiments 1-51, wherein the LFA comprises a target analyte.

Embodiment 54: The device of embodiment 53, wherein the device is configured to provide a sandwich assay when the LFA is used.

Embodiment 55: The device of any one of embodiments 18 to 54, wherein the LFA comprises a probe capture moiety, wherein the probe capture moiety interacts with a probe or a component thereof.

Embodiment 56: The method according to any one of embodiments 1 to 55, wherein the components of the first phase solution and/or the components of the second phase solution are dehydrated on and/or in the LFA strip, and upon addition of the sample, wherein the mixed phase solution is partitioned into a first phase solution and a second phase solution.

Embodiment 57: The device of any of embodiments 1-56, wherein the LFA comprises a well having a volume sufficient to contain a solution selected from the group consisting of: at least a portion of the ATPS; at least a portion of the first phase solution; at least a portion of the second phase solution; a resuspended solution of the target analyte; and combinations thereof.

Embodiment 58: The device of embodiment 57, wherein the sufficient volume is from about 1 nl to about 5 ml.

Statement 59: The device of statement 57, wherein the well is disposed at a location of the LFA selected from corner, distal, center, junction, off-center, and bend.

Statement 60: The device of any of statements 56-58, wherein the well comprises a pad.

Embodiment 61: The device of embodiment 60, wherein the pad is selected from the group consisting of a salt pad, a buffer pad, a filter pad, a surfactant pad, a probe pad, a polymer pad, and combinations thereof.

Statement 62: The device of any of statements 1-61, wherein the LFA is configured according to a structure selected from the structures shown below: Figures 16, 20 and 62-70.

Statement 63: The apparatus of any of statements 1-62, wherein the LFA comprises a plurality of path routes.

Statement 64: The apparatus of any of statements 1-63, wherein the LFA comprises a dry destination.

Embodiment 65: The device of any of embodiments 1-64, wherein the device comprises a working buffer.

Embodiment 66: The device of any of embodiments 1-65, wherein the device comprises a port for administering a sample to the device.

Statement 67: The apparatus of statement 66, wherein the port is connected to the ATPS.

Statement 68: The apparatus of statement 66, wherein the ATPS and the LFA are integrated, and the port is connected to the LFA.

Statement 69: The device of any of statements 66-68, wherein the port comprises a structure selected from the group consisting of a tube, a funnel, a valve, a syringe, a straw, a channel, a plunger, a piston, a pump, and combinations thereof. .

Statement 70: The apparatus of any of statements 1-69, wherein the apparatus does not require a power supply.

Embodiment 71: The device of any of embodiments 1-70, wherein the ATPS and the LFA are integrated prior to use of the device.

Embodiment 72: The device of any of embodiments 1-70, wherein the ATPS and the LFA are separated prior to use of the device.

Embodiment 73: The device of embodiment 72, wherein the device is configured to insert LFA into ATPS.

Embodiment 74: The device of any of embodiments 72 or 73, wherein the device comprises: a: a first component comprising a chamber for containing the ATPS; and b: a second component comprising the LFA.

Embodiment 75: The device of any one of embodiments 1 to 74, wherein the device comprises an actuator that delivers a sample and/or a target analyte to ATPS.

Embodiment 76: The device of any of embodiments 1-75, wherein the device comprises an actuator that delivers a solution to the LFA.

Embodiment 77: The device of embodiment 76, wherein the solution is selected from the group consisting of a mixed phase solution, a first phase solution, a second phase solution, and combinations thereof.

Embodiment 78: The device of any of embodiments 1-77, wherein the ATPS and the LFA are contained in a single housing.

Statement 79: The device of any of statements 1-78, wherein the device is a portable device.

Embodiment 80: A method of detecting and/or quantifying a target analyte in a sample, comprising: a: applying the sample to a device according to any one of embodiments 1 to 78; and b: detecting the presence or absence of the target analyte and/or quantifying the target analyte on the LFA.

Embodiment 81: The method of embodiment 80, wherein the method comprises subjecting the sample to ATPS.

Embodiment 82: The method of embodiment 80, wherein the method comprises applying a sample to an LFA, wherein the LFA and the ATPS are integrated.

Embodiment 83: The method of any one of embodiments 80-82, wherein the method comprises concentrating the target analyte in ATPS.

Embodiment 84: The method of any one of embodiments 80-83, wherein the method comprises concentrating the target analyte in LFA.

Embodiment 85: The method of any one of embodiments 80-84, wherein the sample is selected from the group consisting of a tissue/fluid from a biological organism, a food sample, a chemical sample, a drug sample, an environmental sample, and combinations thereof.

Embodiment 86: The method of any one of embodiments 80-85, wherein the sample is selected from the group consisting of a blood sample, a swab sample, a serum sample, a plasma sample, a urine sample, a saliva sample, and combinations thereof.

Embodiment 87: The method of any one of embodiments 80 to 86, wherein the sample comprises bacteria, viruses, protozoa, algae, fungi, drugs, pathogens, mammals, toxins, environmental pollutants and components thereof, and combinations thereof. from a source selected from the group consisting of

Embodiment 88: The method according to any one of embodiments 80 to 87, wherein the target analyte comprises a biological molecule.

Embodiment 89: The method of embodiment 88, wherein the biological molecule is selected from the group consisting of nucleic acids, proteins, metabolites, lipids, small molecules, sugars, antibodies, antigens, enzymes and combinations thereof.

Embodiment 90: A paper fluidic device for detecting a target analyte in a sample, wherein the paper fluidic device comprises a porous matrix, the porous matrix comprising: a: containing ATPS or a component thereof; b: when said ATPS or a component thereof is present in a fluid phase, said ATPS or component thereof is configured to flow through said porous matrix and has sufficient porosity to allow flow.

Embodiment 91: The paper fluid device of embodiment 90, wherein the device contains ATPS or a component thereof.

Embodiment 92: The method of embodiment 90 or 91, wherein said ATPS or a component thereof is selected from the group consisting of a first phase solution, a second phase solution and a mixed phase solution, wherein the mixed phase solution comprises the first phase solution and the A paper fluid device comprising a mixture of second phase solutions.

Statement 93: The paper fluid device of any of statements 91 or 92, wherein the ATPS or component thereof is dehydrated on/in at least the first portion of the porous matrix.

Statement 94: The paper fluid device of statement 93, wherein the first portion of the porous matrix has a different width than the second portion of the porous matrix.

Embodiment 95: The paper fluid device of embodiment 94, wherein the device is configured such that application of the sample to the device hydrates ATPS, thereby providing ATPS or a component thereof on the fluid.

Embodiment 96: The method of any one of embodiments 92 to 95, further adding a well for containing a solution selected from the mixed phase solution, the first phase solution, the second phase solution, the sample, the probe, and combinations thereof. A paper fluid device comprising:

Statement 97: The paper fluid device of statement 96, wherein the well comprises an actuator for releasing the contents of the well into and/or on the porous matrix.

Embodiment 98: The device of any of Embodiments 96 or 97, wherein the well comprises a pad.

Embodiment 99: The device of embodiment 98, wherein the pad is selected from the group consisting of a salt pad, a buffer pad, a filter pad, a surfactant pad, a probe pad, a polymer pad, and combinations thereof.

Statement 100: The paper fluid device of any of statements 92-99, wherein, in use, the first phase solution and the second phase solution flow through the porous matrix at different rates.

Statement 101: The paper fluid device of any of statements 92-100, wherein, in use, the first phase solution and the second phase solution flow through the porous matrix in different directions.

Statement 102: The paper fluid device of any of statements 92-101, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a polymer.

Statement 103: The paper fluid device of any of statements 92-101, wherein the first phase solution comprises a micellar solution and the second phase solution comprises a salt.

Statement 104: The paper fluid device of any of statements 102 or 103, wherein the micellar solution comprises a surfactant.

Statement 105: The paper fluid device of statement 104, wherein the surfactant is selected from the group consisting of a non-ionic surfactant, a surfactant comprising a zwitter ion, and an ionic surfactant.

Statement 106: The paper fluid device of statement 105, wherein the micellar solution comprises Triton-X.

Statement 107: The paper fluid device of any of statements 93-101, wherein the first phase solution comprises a polymer and the second phase solution comprises a polymer.

Statement 108: The paper fluid device of any of statements 92-101, wherein the first phase solution comprises a polymer and the second phase solution comprises a salt.

Statement 109: The paper fluid device of statement 108, wherein the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate.

Embodiment 110: The paper fluid device of any of Embodiments 92 to 101, wherein the first phase solution is selected from component 1 of Table 1 and the second phase solution is selected from component 2 of Table 1.

Statement 111: The paper fluid device of any of statements 90-110, wherein the device is configured according to a structure selected from the structures shown in Figs. 16, 20, and 62-70.

Statement 112: The paper fluid device of any of statements 90-111, wherein the porous matrix comprises a first pathway and a second pathway.

Statement 113: The paper fluid device of statement 112, wherein the first solution preferentially flows through the first pathway and the second phase solution preferentially flows through the second pathway.

Embodiment 114: The paper fluid device of any one of embodiments 90-113, wherein the device comprises a probe that binds to a target analyte to produce a probe-analyte complex.

Embodiment 115: The paper fluid device of any one of embodiments 90-114, wherein the target analyte is bound to a probe in the probe-analyte complex.

Statement 116: The paper fluid device of any of statements 114 or 115, wherein the probe comprises a magnetic particle.

Embodiment 117: The device of embodiment 116, further comprising a magnetic field oriented to attract the magnetic particle to a portion of the porous matrix, wherein the force of the magnetic field on the magnetic particle is directed toward the portion of the porous matrix of the probe-analyte complex. A paper fluid device that enhances flow.

Embodiment 118: The method of any one of embodiments 114 to 117, wherein the probe is made of polyethylene, polypropylene, nylon, polyoxymethylene, polytetrafluoroethylene (TEFLON®), dextran, polyvinyl chloride, and combinations thereof. A paper fluid device comprising a polymer selected from the group consisting of.

Embodiment 119: The paper fluid device of any of embodiments 114-118, wherein the probe comprises a biological polymer selected from the group consisting of cellulose and chitin.

Statement 120: The paper of any of statements 114-119, wherein the probe comprises a metal selected from the group consisting of gold, silver, titanium, stainless steel, aluminum, platinum and alloys thereof, and combinations thereof. fluid device.

Embodiment 121: The paper fluid device of any of embodiments 114-120, wherein the probe comprises a nanoparticle.

Statement 122: The paper fluid device of statement 121, wherein the nanoparticles are gold nanoparticles.

Statement 123: The paper fluid device of any of statements 114-122, wherein the probe comprises a coating.

Statement 124: The paper fluid device of statement 123, wherein the coating comprises a polymer selected from polypropylene glycol and polyethylene glycol.

Statement 125: The paper fluid device of statement 123, wherein the coating comprises dextran.

Embodiment 126: The paper fluid device of embodiment 123, wherein the coating comprises a hydrophilic protein.

Embodiment 127: The paper fluid device of embodiment 123, wherein the coating comprises serum albumin.

Statement 128: The paper fluid device of any of statements 123-127, wherein the coating has an affinity for the first phase solution or the second phase solution.

Embodiment 129: The paper fluid device of any of embodiments 114-128, wherein the probe comprises a binding moiety that binds to a target analyte.

Embodiment 130: The paper fluid device of embodiment 129, wherein the binding moiety is selected from the group consisting of an antibody, a lectin, a protein, a metabolite, a glycoprotein, a nucleic acid, a small molecule, a polymer, and a lipid.

Embodiment 131: The paper fluid device of embodiment 129, wherein the binding moiety is an antibody or antibody fragment.

Embodiment 132: The paper fluid device of any of embodiments 114-131, wherein the probe comprises a detectable label.

Embodiment 133: The paper fluid device of embodiment 132, wherein the detectable label is selected from the group consisting of a colorimetric label, a fluorescent label, an enzymatic label, a colorigenic label, a radioactive label, and combinations thereof.

Statement 134: The device of any of statements 90-133, wherein the device comprises a dry destination, wherein the first aqueous solution or the second phase solution preferentially flows through a porous matrix towards the dry destination. paper fluid device.

Embodiment 135: The device of any one of Embodiments 90-134, wherein the device comprises a working buffer, wherein the first phase solution and/or the second phase solution more rapidly through the porous matrix upon contact with the working buffer. A flowing, paper fluid device.

Embodiment 136: The material of any one of Embodiments 90-135, wherein the porous matrix is selected from cellulose, glass fibers, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, and combinations thereof. A paper fluid device comprising:

Embodiment 137: The paper fluidic device of any of embodiments 90-136, wherein the porous matrix comprises a probe capture moiety, wherein, in use, the probe capture moiety interacts with the probe or component thereof.

Embodiment 138: The paper fluid of any one of embodiments 90-137, wherein the porous matrix comprises a target analyte capture moiety, wherein in use the target analyte capture moiety interacts with the target analyte or component thereof. Device.

Statement 139: The paper fluid device of statement 138, wherein the LFA is configured to provide a sandwich assay when the paper fluid device is used.

Embodiment 140: The paper fluid device of any of embodiments 90-137, wherein the porous matrix comprises a target analyte.

Statement 141: The paper fluid device of clause 140, wherein the LFA is configured to provide a competition assay when the paper fluid device is used.

Embodiment 142: The paper fluid device of any of embodiments 90-141, wherein the porous matrix is configured to concentrate the target analyte as it flows through the porous matrix.

Embodiment 143: The paper fluid device of any one of embodiments 90-142, wherein the paper fluid device further comprises a control analyte, and wherein comparing the control analyte to the target analyte on a porous matrix provides for quantification of the target analyte. which is a paper fluid device.

Embodiment 144: A method of detecting or quantifying a target analyte in a sample, the method comprising: a: applying the sample to a device according to any one of embodiments 90 to 143; and b: detecting the presence or absence of the target analyte and/or quantifying the target analyte.

Example

The following specific and non-limiting examples are to be construed as illustrative only and do not limit the invention in any way. Without further elaboration, it is believed that one of ordinary skill in the art, based on the description herein, can use the present invention to its fullest extent. All publications cited herein are incorporated herein by reference in their entirety.

Example One. To concentrate magnets or solids in a two-phase system PEGylation gold a nano probe ATPS used

Two approaches were investigated to integrate an aqueous two-phase system (ATPS) with a lateral flow immunoassay (LFA) to enhance the sensitivity of LFA. The first approach used magnets to collect magnetic nanoprobes capable of capturing target proteins from an ATPS solution. A second approach uses a newly discovered phenomenon in which the surface chemistry of nanoprobes has been engineered such that they split at the solid-liquid interface between ATPS and polypropylene surfaces to enable rapid and easy collection without the use of syringes or magnets. did.

In order to efficiently recover magnetic nanoparticles, it has been assumed that the required size of magnetic nanoparticles must be large enough to respond to a magnetic field. However, if the size of the particles is too large, the particles may experience difficulty moving through the LFA test strip.

To address this problem, ATPS together with gold-coated magnetic nanoprobes (GMP) were used to first capture target biomolecules in the sample solution. To avoid using expensive bulky magnets to collect particles from solutions or giant nanoprobes that could respond to weak magnetic fields, ATPS first passively concentrates GMP in one of the two bulk phases of ATPS to small volumes. was used for Subsequently, inexpensive portable magnets were used to rapidly recover GMP and captured biomolecules. Since the gold coating on GMP acts as a colorimetric indicator of the LFA, GMP was applied directly to the LFA strip.

In addition to using ATPS to enhance magnetic extraction of GMP, it was found that phase separation of ATPS benefited from the use of GMP and magnetic forces. The PEG-salt ATPS phase is completely separated in chronological order. Because the concentration of nanoprobes remains constant on the lower PEG-depleted phase as a result of minimal influx of PEG-rich domains on the macroscopic PEG-depleted phase, extraction of concentrated samples may occur before the system reaches equilibrium, but for subsequent LFA Thirty minutes were still required to achieve sufficient volume of the lower phase to be extracted. Alternatively, GMP in ATPS split into PEG-deficient domains extremely almost immediately when phase separation was induced. PEG-deficient domains containing GMP can more readily find each other in the presence of magnetic forces and collide with each other, possibly due to GMP dragging the PEG-deficient domains with them when they respond to a magnetic field. Together with gravity, this force further facilitated the incorporation of the PEG-deficient domains, leading to accelerated phase separation and obtaining sufficient volume of the lower phase for extraction in just 10 minutes.

In this study, a GMP small enough to travel through an LFA test strip without flow problems was first made. This GMP was then used to capture the model protein, transferrin (Tf), in a PEG-salt ATPS solution yielding: 9:1 (upper phase: lower phase) volume ratio. A small magnet (1/8'' diameter x 1/32'' thick) was placed in the solution to promote phase separation. GMP and captured protein were recovered after 10 minutes, the small magnet was removed, and the GMP-Tf complex was directly applied to the LFA for detection.

Although the above approach used ATPS with magnetic nanoprobes to efficiently enrich and detect Tf, a small magnet was still needed. Although the following approach explored the possibility of extracting target-nanoprobe complexes without the use of an external magnetic field, the surface chemistry of pegylated gold nanoprobes (PGNPs) is extremely difficult at the solid-liquid interface between ATPS and polypropylene (PP) surfaces. was engineered to be divided into This unique phenomenon was found to be ATPS-specific, as PGNPs did not resolve at the solid-liquid interface when aqueous solutions composed only of polymers or salts were used. In addition, this phenomenon was also found to be material-specific, as PGNPs are extremely cleaved at the polypropylene (PP) surface in the present ATPS and not at the glass surface.

To facilitate the collection of the target-PGNP complex as a PP-ATPS solid-liquid interface, PGNP decorated with anti-Tf antibody was added to the ATPS solution containing Tf, and then a commercially available PP straw was submerged in ATPS. PGNP first captures the target protein on ATPS and then preferentially cleaves on the PP surface. After 10 min, the PP straws were removed from ATPS with significant amounts of protein-GNP complexes still adsorbed to the surface. This effectively eliminated the need to manually extract the concentrated Tf-PGNP using a syringe.

In this study, PGNP was cleaved at the PP-liquid interface in ATPS. This PGNP was then used to capture Tf in a PEG-salt ATPS solution yielding a 1:1 volume ratio. The PP straw was immersed in ATPS solution, and PGNP and captured protein were recovered after 10 minutes by pulling the straw from the solution. The PGNP-Tf complex on the straw was washed with running buffer and applied directly to LFA for detection. Due to the minimal volume associated with the PP-ATPS interface and the small volume required to wash off the PGNP from the straw, the PGNP was extremely concentrated and the detection limit of Tf in LFA was improved by a factor of 50.

To prepare LFA strips, an LFA test using a competitive mechanism was performed. In a competition assay, the target of interest is immobilized on a nitrocellulose membrane to form a test line. An immobilized secondary antibody to the primary antibody on the nanoprobe (GMP or PGNP) constitutes the control line. In LFA, when the sample first contacts the nanoprobes, if target molecules are present in the sample, they bind to their specific antibody decorated on the nanoprobe. Once the target molecules present in the sample saturate the antibody on the nanoprobes, these nanoprobes can no longer bind the immobilized target molecules on the test strip. As a result, the nanoprobe did not form a visible band in the test line, indicating a positive result. On the other hand, if the sample does not contain the target molecule at a concentration capable of saturating the antibody on the nanoprobe, such antibody on the nanoprobe can bind to the immobilized target molecule on the test strip and form a visual band in the test line. This indicates a negative result. Also, regardless of the presence of the target molecule in the sample, the antibody on the nanoprobe binds to the immobilized secondary antibody on the control line, indicating that sufficient sample has wicked through the test line to reach the control line. The presence of a visible control line indicates a validated test.

To prepare GNPs, iron oxide magnet nanoparticles with a diameter of 50 nm were coated with gold. Briefly, iron oxide particles were diluted with 0.01 M sodium citrate and stirred for 10 minutes. Subsequently, four additions of 1% w/v gold chloride solution were applied to the iron oxide nanoparticle solution with stirring at 10-minute intervals. The solution changed from black to brown after addition of gold chloride. The gold-coated magnetic nanoparticles were then recovered using a magnet.

The collector was built by placing a small neodymium magnet (1/8'' diameter x 1/32'' thick, KJ Magnetics, Philadelphia, PA) at the end of a drinking straw. The magnets were wrapped in parafilm to avoid direct contact with the GMP in solution.

To ensure that GMP is extremely cleaved on the bottom of the PEG-salt ATPS, on the PEG-deficient phase, 30 μL of GMP was added to the 2mL ATPS solution obtained in a 9:1 volume ratio. To determine whether the presence of a magnet promotes phase separation, splitting experiments were performed with or without a magnetic collector. The volume of the lower phase was measured for each experiment at 10 and 30 minutes.

It was then tested whether GMP could be introduced with ATPS and LFA. First, 2 mL of a 9:1 ATPS solution spiked with various concentrations of Tf was prepared. 30 μL of GMP was applied to each solution, then a magnetic collector was added. The solution was placed in a 25 ^o C water bath and incubated for 10 minutes. After the incubation period, the collector was removed from the solution and transferred to a tube containing 50 μL of working buffer. The small magnet was removed from the straw, and the GMP collected on the straw was washed away by shaking the straw in working buffer for a few seconds. The straws were then removed and the LFA test strips were immersed in working buffer containing the collected GMP. After 10 minutes, the test strips were removed from the tube and images of the test strips were taken using a Canon EOS 1000D camera (Canon USA, Inc., Lake Success, NY).

Exposed gold nanoparticles were prepared to produce a clear cherry-colored solution with a particle size of about 25-30 nm in diameter. To prepare PGNP, 320 mg of goat anti-Tf antibody was incubated in 20 mL of colloidal gold solution for 30 min, followed by addition of thiolated-PEG2000 using a molar ratio of 5000:1 to: PEG: NPs and an additional incubation of 30 min. To prevent non-specific binding of other proteins to the surface of colloidal gold, 2 mL of a 10% bovine serum albumin (BSA) solution was added to the mixture and mixed for an additional 15 minutes. The resulting solution was gently mixed on a shaker during the incubation period. To remove free unbound antibody, PEG and BSA, the mixture was subsequently centrifuged at 4 ^° C and 9,000 g for 30 min. The pellet of PGNP was washed with 1% BSA solution, and this washing step was repeated twice. Finally, the recovered PGNP was resuspended in 3 mL of 0.1M sodium borate buffer at pH 9.0.

A 1:1 volume ratio was obtained and PEG-salt ATPS solutions containing varying Tf concentrations were prepared first in glass tubes. 5 μL of GNP solution was added to the Tf-ATPS solution to a final volume of 1 mL. The ATPS solution was thoroughly mixed, and a PP straw was inserted into each ATPS solution. The solution was then incubated at 25 ^° C for 15 min. For each mixture, the PP straw was carefully removed from the ATPS solution. Assuming the total volume of the PP-ATPS interface was approximately 1 µL, 49 µL of test buffer was used to wash the adsorbed Tf-GNP complex from the portion of the straw soaked in ATPS solution to achieve a total volume of 50 µL. Both the inner and outer surfaces of the straw were washed for a total of 10 seconds. The LFA test strips were then immersed vertically so that the sample pad was in contact with the mixture. After 10 minutes, the strips were withdrawn from the mixture and images of the test strips were immediately taken using a Canon EOS 1000D camera (Canon, USA, Inc., Lake Success, NY).

The GMP developed in this study achieved four functions: (1) capture the target protein in the sample; (2) extremely split in one of the bulk phases of ATPS; (3) being collected by inexpensive portable magnets; (4) After being extracted and applied to the LFA paper strip, it functions as a colorimetric indicator for LFA directly. Functions (2) and (3) were demonstrated by placing GMP in a PEG-salt ATPS solution. As shown in: FIG. 21A , it is observed that GMP is extremely cleaved in the lower PEG-deficient phase, due to the repulsive steric exclusion-volume interaction operating between the GMP and the greater number of PEG molecules in the PEG-rich phase. became This made it possible to concentrate GMPs in small volumes, which enabled their collection using much smaller and lighter magnets ( Fig. 21b ). In addition, since these GMPs were highly reactive when they were present in close proximity to the magnet, the particles could be quickly recovered and transferred for subsequent detection ( FIG. 21C ). Finally, by using this approach, GMP could be collected in very small volumes, which enabled extreme concentrations of the target protein.

To confirm whether GMP could enhance the sensitivity of LFA when used in combination with ATPS, a preliminary LFA study with or without a pre-concentration step was performed ( FIG. 22 ).

A second approach for integrating ATPS and LFA was to drive pegylated gold nanoprobes (PGNPs) at the solid-liquid interface between ATPS and polypropylene (PP) solids. Similar to the first approach, but this approach no longer requires an external magnet. This mechanism requires ATPS. As shown in Figure 23a and b , PGNP did not resolve at the solid-liquid interface when either PEG alone or aqueous salt alone solution was used. Indeed, it has been shown that PGNPs are uniformly dispersed throughout each aqueous solution with no preference for a solid-liquid interface. Since PGNP was found to split at the solid-liquid interface only in the presence of phase-separated ATPS, ATPS with a 1:1 volume ratio was used in this study because the ATPS phase separates the fastest when compared to ATPS with other volume ratios. became 23C demonstrates the extreme splitting behavior of PGNPs on PP straws after 10 min. The PP straw also allowed for easy extraction of the concentrated target-PGNP complex. After the straw was withdrawn, the collected PGNPs were easily washed off using working buffer and applied directly to LFA ( FIG. 23D ).

The LFA results are shown in FIG . 24 . Each test indicated that the fluid had completely flowed through the strip and indicated the presence of a control line confirming the validity of the test. For the negative control in the absence of Tf, a test line was shown and thus a true negative result. At high Tf concentrations (10 ng/μL), no test line appeared, thus giving a true positive result. However, at low Tf concentrations in the absence of a prior enrichment step, a visible test line appeared suggesting that an insufficient amount of Tf was present in the sample to saturate the antibody decorated on PGNP. Therefore, these tests gave false negative results. LFA results using this approach are shown in the top panel ( FIG. 24 top). As false negative results were found at 1 ng/μL in the LFA study without prior enrichment, this indicated that the detection limit of LFA for Tf was 10 ng/μL.

To improve the performance of LFA, the same amount of PGNP used in the single LFA test was placed in ATPS solution with a Tf concentration of 1 ng/μL. By increasing the volume of this ATPS solution by 50-fold compared to the volume used for the LFA strip, PGNPs were able to interact with 50-fold more Tf, thus achieving a high level of saturation. However, since PGNPs were also diluted 50-fold, they need to be concentrated and collected prior to detection. By placing the PP straw in the ATPS solution, the PGNP was extremely split at the surface of the straw. After the ATPS solution was incubated at room temperature for 10 min, the PP straw was removed from the solution. Unlike other adsorption phenomena in which material on a solid surface is typically not recovered, PGNP was readily recovered by washing the straw with a working buffer. This buffer containing PGNP was then applied directly to the LFA. LFA results using this approach are shown in the lower panel ( Figure 24 lower). Since 0.2 ng/μL is the lowest concentration demonstrating a truly positive result, the detection limit of LFA for Tf improved with a 50-fold improvement from 10 ng/μL to 0.2 ng/μL. The portable device uses these two approaches and allows the user to operate the assay with minimal steps ( FIG. 25 ).

Example 2 ATPS and dextran coated gold nanoparticles

Traditional citrate capped or exposed gold nanoparticles typically do not aggregate in aqueous solution due to electrostatic repulsive interactions generated from the negative charge of surface citrate ions. However, they aggregate in solutions of high ionic strength, where van der Waals attraction interactions are greater than electrostatic repulsion interactions. Dextran coated gold nanoparticles were successfully prepared by using dextran instead of citrate as both a reducing agent and a stabilizer. The stability of dextran-coated gold nanoparticles was compared with traditional citrate-capped gold nanoparticles and conventionally used PEG-coated gold nanoparticles. The phase separation behavior of PEG-salt ATPS was optimized to enable rapid extraction of DGNPs at room temperature. The cleavage behavior of DGNP was subsequently investigated in PEG-salt ATPS. Transferrin (Tf), a common serum protein for iron transport, was chosen as a model protein biomarker. The detection limit of LFA for Tf was determined with or without a pre-enriched ATPS step using DGNP. Qualitative results of LFA were interpreted visually, and computer image analysis was also performed for further quantitative confirmation of LFA results.

Traditional citrate capped or exposed gold nanoparticles were prepared. Briefly, 10 mL of ddH 2 O was heated to 100 ^° C with stirring. When the hot pig solution started boiling, 100 μL of 1% w/v gold(III) chloride solution was added. The solution was stirred and boiled for 1 minute, then 25 μL of 6% w/v sodium citrate solution was added. The solution quickly changed from dark purple to clear and dark cherry color, after which it was cooled to room temperature and stored at 4 ^° C.

Prior to preparing the PEG-coated (PEGylated) gold nanoparticles, the concentration of the previously prepared exposed gold nanoparticle solution was determined. This concentration used to determine the amount of PEG required for any given PEG:gold nanoparticle molar ratio was calculated using Beer's Law with a path length of 1 cm:

where C is the concentration of gold nanoparticles in molar concentration units, A is the peak absorbance value of the gold nanoparticles, ε is the molar extinction coefficient in units of M ⁻¹ cm ⁻¹ , and l is the path length in cm . To determine the ε value, the diameter of the nanoparticles was first quantified by dynamic light scattering (DLS) using a Zetasizer Nano ZS particle analyzer (Malvern Instruments Inc, Westborough, Massachusetts). The molar extinction coefficient was then read from a data sheet provided by BBInternational Life Sciences (Madison, WI), which correlated the ε value with the diameter of the gold nanoparticles. The peak absorbance values of the gold nanoparticles were then quantified using a UV-visible spectrophotometer (Thermo Fisher Scientific, Madison, Wisconsin), and the concentration was determined using: Eq. (One).

Methoxy-polyethylene glycol (PEG)-thiol (MW 5000, Nanocs Inc., New York, NY) was used to decorate the surface of gold nanoparticles with a PEG layer. Briefly, 5 mL of a solution of exposed gold nanoparticles was first adjusted to pH 9.0 using 1N NaOH solution. Methoxy-PEG-thiol was added to the particle solution as follows: 50,000:1 PEG:nanoparticle molar ratio to ensure that the gold nanoparticle surface was fully coated. The mixture was then reacted at room temperature for 30 minutes to allow the thiol groups on the PEG to form coordination bonds with the gold nanoparticles. Free PEG was removed by centrifugation at 9000 g for 30 min. The resulting pegylated gold nanoparticles were resuspended in 500 μL of ddH ₂ O.

Dextran coated gold nanoparticles were prepared. Briefly, 12 g of dextran (MW 15,000 - 25,000) is dissolved in 160 mL of ultrapure sterile water (Rockland Immunochemicals Inc., Gilbertsville, PA), and the solution is heated to 100 ^° C with stirring. When the heated solution started boiling, 2.16 mL of 1% w/v gold(III) chloride solution was added. The solution was stirred and boiled for 20 minutes until the color of the solution turned purple. The solution was then cooled to room temperature and stored at 4 ^° C. Excess dextran was subsequently removed by centrifugation at 9000 g for 30 minutes.

The exposed ( ie , citrate capped), pegylated and dextran coated gold nanoparticle solutions were mixed separately with various concentrations of the stock potassium phosphate solution. The mixed solution was incubated at room temperature for 30 minutes. The absorbance spectrum of each sample was determined using a UV-visible spectrophotometer. Critical aggregation concentration (CCC) was identified as the salt concentration at which the absorbance spectrum of the GNP solution near the peak absorbance wavelength significantly decreased in intensity.

Stock potassium phosphate solution (5:1 dibasic: monobasic) was prepared in Dulbecco's phosphate buffered saline (PBS) (Invitrogen, pH 7.4, 1.47 mM KH ₂ PO ₄ , 8.10 mM Na ₂ HPO ₄ , 138 mM NaCl, 2.67). mM KCl and 0.495 mM MgCl ₂ ). Subsequently, a solution of sutok polyethylene glycol (MW 8000, VWR, Brisbane, CA) was prepared in PBS. Using the stock solution, 2 mL PEG-salt ATPS solutions in PBS were prepared at specific concentrations of PEG and salt yielding a PEG rich (salt-poor):PEG-deficient (salt rich) volume ratio of 9:1. This volume ratio was determined after the ATPS solution was incubated at room temperature for at least 12 hours.

Iodine-125 ( ¹²⁵ I) was used to radiolabel the tyrosine residue of a goat anti-Tf antibody (Bethyl Laboratories, Montgomery, TX). Briefly, Na ¹²⁵ I (MP Biomedicals, Irvine, CA) was activated with IODO-BEADS (Pierce Biotechnology, Rockford, IL). Subsequently, activated ¹²⁵ I was reacted with goat anti-Tf antibody for 15 min. Radiolabeled antibody was purified from free ¹²⁵ I using a Sephadex G15 size exclusion column. The specific activity and concentration of the radiolabeled antibody was determined by a phosphotenstic acid assay.

To prepare dextran-coated gold nanoprobes (DGNPs), 10 mL of previously prepared dextran-coated gold nanoparticle solution was first adjusted to pH 9.0 using 1N NaOH. Subsequently, 80 μg of goat anti-Tf antibody was added to the dextran coated gold nanoparticle solution. The mixture was then reacted at room temperature for 30 minutes to allow the antibody to form coordinate bonds with the particles. Free unbound antibody was removed by centrifugation at 9000 g for 30 min. The recovered DGNPs were further washed with 1% bovine serum albumin (BSA) solution to allow BSA to coat any glass surface on DGNPs to prevent non-specific binding. Excess BSA and additional unbound antibody were removed by centrifugation at 9000 g for 30 min. The recovered DGNP was resuspended in 1 mL of 0.1M sodium borate buffer (pH 9.0).

To study the cleavage behavior of DGNP in PEG-salt ATPS, the concentration of DGNP in each phase of the ATPS solution was quantified. Radiolabeled DGNPs were prepared by conjugating radiolabeled goat anti-Tf antibody onto dextran-coated gold nanoparticles. For each split experiment, 200 μL of radiolabeled DGNP solution was added to 3 identical solutions of PEG-salt ATPS in PBS to a final volume of 2 ml each. The mixture was incubated at 25° C. for 30 min, 2 h, 4 h and 12 h. The PEG-rich salt-poor upper phase and the PEG-rich salt-rich lower phase were carefully extracted and the volume of each phase was measured. The amount of radiolabeled DGNP in each phase was quantified by measuring the radioactivity of the radiolabeled antibody conjugated to DGNP using a Cobra Series Auto-Gamma Counter.

Competitive LFA was used in this study, a detailed schematic can be found in FIG. 27 . First, the Tf protein was immobilized on a nitrocellulose membrane to form a test line. A secondary anti-IgG antibody capable of binding the primary antibody on DGNP was also immobilized downstream of the immobilized Tf protein to form a control line. If the Tf protein is not present in the sample solution, DGNP binds to the Tf printed on the strip, forming a visible band in the test line. On the other hand, when Tf proteins are present in the sample, they first bind their specific antibodies on the surface of DGNPs. When DGNP is saturated with Tf protein, DGNP bypasses the printed Tf on the strip, leading to the absence of a visible band in the test line. Excess DGNP still bound to the control line, indicating that the fluid actually flowed through the strip and the results are valid. In summary, the negative test was indicated by two visible bands, one in the test line and one in the control line, whereas the positive test was indicated by only one visible band in the control line.

LFA without PEG-salt ATPS pre-concentration step was performed prior to combination with ATPS. In this study, PBS solutions containing varying concentrations of Tf were prepared, and each Tf concentration was tested in triplicate. For each LFA strip, 10 μL of the corresponding Tf solution was added to 30 μL of test buffer (0.2% BSA, 0.3% Tween20, 0.2% sodium azide, 0.1% polyethylene glycol) used to aid the flow of the sample through the test strip. , 0.1 M Trizma base, pH 8) and 10 μL of DGNP solution. An LFA test strip was immersed vertically into each mixture so that the sample pad was in contact with the mixture. After 10 minutes, the strips were withdrawn from the mixture and the results were read visually and quantified using MATLAB.

LFA with a PEG-salt ATPS pre-concentration step was subsequently performed. In these experiments, ATPS solutions containing variable Tf concentrations were first prepared, and each Tf concentration was tested in triplicate. Since the volume of the lower phase was about 200 μL, 100 μL of DGNP solution was added to the Tf/ATPS solution to a final volume of 2 mL to achieve a similar DGNP concentration as when the LFA test was performed without the ATPS step. The ATPS solution was thoroughly mixed, and incubated at 25 ^o C for 30 minutes. For each mixture, 20 μL of the PEG-deficient lower phase was extracted and mixed with 30 μL of test buffer to match the volume used in the study without the ATPS step. The LFA test strips were immersed vertically so that the sample pad was in contact with the mixture. After 10 minutes, the strips were withdrawn from the mixture and the results were read visually and quantified using MATLAB.

A custom MATLAB script was written to quantitatively analyze the resulting images. Immediately after the LFA testing was completed, images of the test strips were taken using a Canon EOS 1000D camera (Canon U.S.A., Inc., Lake Success, NY). To ensure consistency between images, lighting was adjusted and each strip was oriented in the same way. This image was cropped and converted to an 8-bit grayscale matrix. Intensities were averaged along an axis perpendicular to the flow and parallel to the test and control lines, and a one-dimensional intensity map was generated. The two maxima were identified as control and test lines, and the distance between these two lines was corrected using negative controls (with strong test and control lines).

To obtain test line intensities from a set of sample data, the positions of the control lines were identified, and the test line positions were determined using pre-calibrated distances. The test line area was set as a 15 pixel-wide area centered on this test line location. The criterion for this measurement was then determined by averaging the signals from two 25 pixel-wide regions located on either side of the test line. The first reference region started 25 pixels upstream of the test line location and continued 25 pixels downstream, while the remainder started 25 pixels downstream of the test line location and continued 25 pixels downstream. The test line intensity was then calculated as the area under the curve of the test line area to capture the effect of any DGNP bound to the test line.

Critical aggregation constant (CCC) values of exposed, PEGylated and dextran-coated gold nanoparticle samples were measured to determine whether the dextran coating provides the required steric stability to gold nanoparticles in a PEG-salt ATPS solution. did. The peak absorbance wavelength of the absorbance spectrum of the gold nanoparticles was about 535 nm. Therefore, the absorbance spectrum around 535 nm for each type of gold nanoparticles was observed over the potassium phosphate concentration range, because calcium phosphate is the salt component of the PEG-salt ATPS. The results are shown in FIG . 28 . For each plot, the peak absorbance value was assigned a value of 1.0 (or 100% normalized absorbance), and the remaining absorbance values of the plot were adjusted accordingly. From this plot, the CCC of the exposed gold nanoparticles was 0.3 to 0.4% w/w potassium phosphate; The CCC of the pegylated gold nanoparticles was 7.5 to 10% w/w potassium phosphate; It was determined that the CCC of the dextran coated gold nanoparticles was greater than 30% w/w potassium phosphate. These results indicated that dextran-coated gold nanoparticles were more stable than even pegylated gold nanoparticles in potassium phosphate salt solution. In addition, since the 30% w/w potassium phosphate is greater than the lower phase potassium phosphate concentration (about 10% w/w), it is expected that the dextran-coated gold nanoparticles will remain stable and functionally in the present PEG-salt ATPS solution. became This has been confirmed by their successful use in the studies described herein.

The cleavage behavior of radiolabeled DGNPs was first visually observed in a PEG-salt ATPS solution containing: 9:1 PEG rich: PEG depleted volume ratio. Initially, the PEG-salt ATPS solution containing DGNP was opaque and displayed a pale purple color ( FIG. 29A ). A lower phase, which was then dark purple, appears, indicating that DGNP is extremely cleaved in the PEG-deficient subphase ( FIG. 29B ). Over time, the lower phase increased in volume as the DGNP-rich, PEG-deficient domains migrated to the bottom of the ATPS solution. In contrast, DGNP showed minimal cleavage in the upper PEG-rich phase when the upper phase had a very slight purple color. After a 12 h equilibration time, the volumes of the upper and lower phases were no longer changed, and the upper phase appeared clear, suggesting that all DGNP rich, PEG deficient domains reached the bottom of the ATPS solution ( FIG. 29C ). After about 30 minutes, the color intensity of the lower phase did not change over time, suggesting that the DGNP concentration in the lower phase remained relatively constant for 30 minutes to 12 hours.

Then, the partitioning behavior of radiolabeled DGNP between the two phases was quantified by the partitioning coefficient (K _DGNP ) defined below:

where C _DGNP , upper and C _DGNP , lower are the concentrations of DGNP in upper and lower phases, respectively. The K _DGNP value was determined to be 0.00605 ± 0.00010, consistent with the idea that relatively large DGNPs should be extremely partitioned in the PEG-deficient subphase.

To demonstrate that DGNP can be enriched in PEG-salt ATPS in a predictive manner, the concentration coefficient, or fold improvement of DGNP, was determined in a 9:1 PEG-salt ATPS solution. The concentration coefficient is defined as the initial DGNP concentration, C _DGNP , the concentration of DGNP in the lower phase divided by the initial, C _DGNP , the lower.

The expression of the concentration coefficient can be derived by starting with the molar balance equation for DGNP in ATPS solution:

Here, V upper and V lower are the volumes of the upper and lower phases, respectively. Combining the following equations: (2) and (3), rearranging the following equations: (4), we obtain:

Since the partitioning coefficient of DGNP is smaller than V lower/V upper, the concentration factor can be further simplified as follows:

This means that the concentration coefficient is only a function of the volume ratio for the extreme K _DGNP values. Thus, with a volume ratio of 9:1, or a lower phase volume that is 1/10 of the volume of the total solution, a concentration factor of 10 is expected. This was confirmed by a split experiment on DGNP, where C _DGNP , lower was measured after 12 hours of incubation. However, since it is undesirable to wait 12 h for ATPS to achieve macroscopic phase separation equilibrium in the control point application, and because the DGNP concentration in the lower phase was time-independent after 30 min, the concentration factor was was measured in 30 shows that the concentration coefficient in ATPS solution remains relatively constant for 30 minutes to 12 hours. As a result, the lower phase was extracted at 30 min for LFA application.

To demonstrate that DGNP can be used as a colorimetric indicator for LFA, LFA was performed for the detection of Tf without a prior concentration ATPS step. The results of this study are shown in FIG . 31A . Since a band was observed on the LFA strip, it was concluded that DGNP was functional as a colorimetric indicator. Each test showed the presence of a control line indicating that the post had completely flowed through the strip and validated the test. For the negative control in which no Tf was present, the appearance of the test line indicated a truly negative result. When high Tf concentrations were used (3.16 ng/μL and 1.0 ng/μL), the test line was absent, giving true positive results. However, at concentrations as low as 0.316 ng/μL, a visible test line appeared, thus indicating a false negative result. This suggested that the detection limit of LFA for Tf without a prior concentration step was 1 ng/μL.

A 9:1 PEG-salt volume ratio solution was used to demonstrate that the detection limit of LFA could be improved 10-fold by concentrating DGNP saturated with Tf. The results of this study are shown in FIG . 31B . After incubating the ATPS solution at room temperature for 30 minutes, the concentrated DGNP in the PEG-deficient lower phase was extracted and applied to LFA. Similar to the previous experiment, both control and test lines appeared in the absence of Tf, indicating valid negative results. While no test line appeared at high Tf concentrations (0.316 ng/μL and 0.1 ng/μL), the visible test line first appeared at 0.0316 ng/μL, with a detection limit of 0.1 for LFA for Tf with a pre-enrichment step. ng/μL. This corresponded to a 10-fold improvement in the detection limit of LFA when DGNP and PEG-salt ATPS were used.

To further confirm the visual interpretation of the LFA results, images of the test strips were taken and the strength of the test lines was quantified. If a pre-enrichment step was used, the test line signal for each LFA was weaker when testing the same Tf concentration, indicating that Tf was enriched and saturated DGNP, where the saturated DGNP was unable to bind to the test line, resulting in a positive result. indicates Both control line and test line signal intensities were similar to negative controls with and without ATPS. This indicated that the amount of DGNP running on the LFA strips in both experiments was similar. MATLAB analysis of the LFA panel also demonstrated an improvement in detection limits ( FIG. 32 ). Specifically, the two curves of FIG. 32 are separated by a 10-fold difference in the initial Tf concentration for approximately the same test line signal.

Example 3. Direct addition of mixed PEG-salt ATPS to LFA

In this example, an all-in-one device that includes both next-generation sensitivity, speed and ease of use, ATPS phase separation and downstream detection functions was optimized. Instead of applying a concentrated sample after phase separation of ATPS, direct addition of mixed ATPS to a paper-based device became possible. The solution will flow towards it's detection area. Separation into its two phases allows the concentration and detection steps to occur simultaneously, further reducing the overall time-to-result. It is proposed that the paper membrane accelerates the macroscopic phase separation of ATPS. To further exploit this phenomenon, the paper apparatus was vertically expanded to increase the flow cross-sectional area, and the effect of capillary action and/or gravity on macroscopic separation was used. In addition to promoting phase separation, this 3-D component also has the ability to handle larger and thinner volumes of sample leading to greater fold improvement. The novel ATPS and LFA in 3-D paper structures successfully yielded a 10-fold improvement in the detection limit of the model protein Tf while reducing overall time-to-results and maintaining ease of use. This device offers significant improvements over traditional LFA assays and can be modified to detect a variety of diseases with low characteristic biomarker levels. This new platform technology is highly sensitive, low cost, fast and equipment free, and thus has the potential to revolutionize the current state of diagnostic medicine within resource scarce areas.

Determination of the polymer-salt ATPS solution volume ratio

All materials, chemicals and reagents, unless otherwise noted, were purchased from Sigma-Aldrich (St. Louis, MO). Polyethylene glycol 8000 (PEG, VWR, Brisbane, CA) and potassium phosphate salt (5:1 dibasic to monobasic ratio) were mixed with Dulbecco's phosphate buffered saline (PBS; Invitrogen, Grand Island, NY, pH 7.4, 1.47 mM). KH ₂ PO ₄ , including 8.1 mM Na ₂ HPO ₄ , 137.92 mM NaCl, 2.67 mM KCl, and 0.49 mM MgCl ₂ ). Equilibrium volume ratios (volume of upper phase divided by volume of lower phase) were obtained by varying the w/w composition of PEG and salt along the same tie line. 1:1 and 9:1 volume ratio ATPS were found and used in further experiments.

Preparation of antibody-decorated dextran-coated gold nanoprobes (DGNPs)

Dextran-coated gold nanoparticles were synthesized. Briefly , 6 g of dextran (Mw. Leuconostoc spp ) ) were dissolved in 80 mL of filtered ultrapure sterile water (Rockland Immunochemicals Inc., Gilbertsville, PA). After the solution was stirred and heated to boiling, 1080 μL of 1% w/v gold(III) chloride hydrate solution was added. The color of the reaction mixture changed to reddish purple, stirred and boiled for about 20 minutes. The newly formed dextran-coated gold nanoparticles were centrifuged to remove free dextran and resuspended in 70 mL of water. To form functionalized DGNPs, the pH of the dextran coated gold nanoparticle solution was adjusted to 9.0 using 1.5M NaOH. For every ml of dextran-coated gold nanoparticle solution, 8 μg of anti-Tf antibody (Bethyl Laboratories, Montgomery, TX) was added. The reaction mixture was placed on a shaker for 30 minutes to promote the formation of coordination bonds between the antibody and the dextran coated gold nanoparticles. Free antibody was removed by centrifugation. The pellet was resuspended in 100 μL of 0.1M sodium borate buffer at pH 9.0.

ATPS visualization

To visualize the two phases of ATPS, dextran-coated gold nanoparticles, which are purple due to surface plasmon resonance, and brilliant blue FCF dye (The Kroger Co., Cincinnati, OH) were mixed in 1:1 and 9:1 volume ratios. 3 g of a total PBS solution containing a predetermined concentration of PEG and salt was taken. These solutions were thoroughly mixed by vortexing and incubated at 25 °C. A picture of the solution was taken when ATPS reached equilibrium. All images were captured using a Canon EOS 1000D camera (Canon U.S.A., Inc., Lake Success, NY).

The two phases of ATPS were then visualized as they flowed along the paper membrane. Two 8 x 30 mm strips of glass fiber paper were laser cut using a VersaLASER 3.50 (Universal Laser Systems, Scottsdale, AZ). Subsequently, 50 mg of mixed ATPS containing Brilliant Blue FCF dye and dextran coated gold nanoparticles (corresponding to a 1:1 or 9:1 equilibrium volume ratio) was added dropwise to one end of the strip using a pipette. Images of the resulting flow were captured at 0 s, 30 s, 105 s and 300 s. The video was also shot with the 8-megapixel camera of a typical smartphone (Apple Inc., Cupertino, CA).

To visualize the phase separation of ATPS in 3-D paper wells, 140 mg of mixed ATPS containing brilliant blue FCF dye and dextran-coated gold nanoparticles were added to the paper wells. A 3-D paper well was formed by laminating nine 8 x 10 mm laser cut strips of glass fiber over one edge of an 8 x 60 mm laser cut strip of glass fiber. After mixed ATPS was applied to the 3-D paper wells, 50 μL of working buffer (0.2% bovine serum albumin (BSA), 0.3% Tween20, 0.1M Trizma base, pH 8) was added to the 3-D paper wells. A working buffer was added to aid the flow of the sample from the paper wells to the rest of the device. Video was taken and images were captured at 0 and 30 seconds, upon addition of the running buffer and after completion of flow.

Detection of Tf

LFA test for detection of Tf

LFA test strips using a competitive assay format were assembled. Briefly, DGNPs decorated with anti-Tf antibody were first added to the sample solution and allowed to bind to any Tf present in the sample to form a DGNP/Tf complex. To confirm the detection limit of Tf with LFA, 30 μL of working buffer and 20 μL of a sample solution consisting of 15 μL of known amount of Tf and 5 μL of DGNP in PBS were mixed in vitro. The LFA test strip was inserted vertically into the tube with the sample pad submerged in the sample, and the fluid wicked through the strip towards the absorbent pad. In the presence of Tf, the DGNP/Tf complex migrating through the LFA strip could not bind to the Tf immobilized in the test line, giving a positive result with the presence of a single band in the control line. Alternatively, if Tf is not present, the antibody on DGNP can bind to Tf on the test line. Since these probes exhibit a reddish-purple color, a visible band is formed when DGNP accumulates in the test line, indicating a negative result. Regardless of the presence of Tf, the antibody on DGNP always binds to the secondary antibody immobilized on the control line. A band in the control line means that the sample flowed completely through the strip indicating a valid test. Thus, a negative result is represented by two bands: one in the test line and one in the control line. In contrast, a positive result appears as a single band in the control line. Each Tf concentration was tested in triplicate. Representative LFA strips were imaged with a Canon EOS 1000D camera in a controlled light condition after 10 min.

Detection of Tf Using 3-D Paper Wells

The LFA component of the paper-based device was slightly modified from the setup described above. Specifically, the cellulosic sample pad was exchanged with 5 x 20 mm glass fiber paper connected to a nitrocellulose membrane containing test and control lines. At the initiation of the sample pad, a 3-D paper well composed of multiple strips of glass fibers was used. For experiments using 1:1 volume ratio ATPS, the wells consisted of 5 (4 5 x 7 mm strips + lower sample pad) layers of glass fiber paper. To start the test, 40 μL of a mixed 1:1 volume ratio ATPS containing a known concentration of Tf was added to the paper wells followed by the addition of 50 μL of working buffer. Images were captured 10 minutes later. For experiments using a 9:1 volume ratio ATPS, 20 layers of paper (19 5 x 7 mm strips + bottom sample pad) were used to form the paper wells. 200 μL of mixed 9:1 volume ratio ATPS containing a known concentration of Tf was added to the paper wells, incubated for 10 minutes, and then 100 μL of working buffer was added. After another 10 minutes, images were captured with a Canon EOS 1000D camera under a controlled lighting environment. Each Tf concentration was tested in triplicate.

ATPS visualization

Integration of ATPS and LFA could significantly improve the detection limit of traditional LFA without sacrificing its advantages. To achieve this, it was first necessary to identify ATPS, whose phases could be visualized as the paper flows through the membrane. Specifically, PEG-salt ATPS was used, which formed a more hydrophobic PEG-rich phase on top and a more dense and hydrophilic PEG-deficient phase on the bottom. Biomolecular cleavage in ATPS is primarily due to relative hydrophilicity (since biomolecules tend to favor the phase in which they experience the highest attractive interaction) and size (larger biomolecules typically experience with a greater number of PEG molecules in the PEG rich phase). (since it does not remain in the PEG abundance phase due to larger steric exclusion-volume repulsion interactions). Brilliant Blue FCF dye is added to the mixed ATPS, and because it is small and hydrophobic, the dye is extremely cleaved in the PEG rich phase. Purple dextran coated gold nanoparticles are also added to the mixed ATPS, and because they are large (about 50 nm diameter measured by dynamic light scattering) and hydrophilic, they are extremely resolved on PEG deficiency. Images of 1:1 volume ratio ATPS and images of 9:1 volume ratio ATPS were taken before and after phase separation ( FIG. 33 ). The amount of Brilliant Blue FCF and dextran coated gold nanoparticles was kept constant between 1:1 and 9:1 volume ratio ATPS. As a result, after phase separation, the upper phase of the 9:1 volume ratio ATPS had a larger volume and thus less concentrated with blue dye compared to the upper phase of the 1:1 volume ratio ATPS. In addition, the lower phase of the 9:1 volume ratio ATPS became smaller in volume, and shrinking the lower phase by displaying a darker purple shade than the 1:1 volume ratio ATPS effectively enriched the dextran-coated gold nanoparticles within the ATPS. prove that it can be done A 9:1 volume ratio ATPS sample was expected to concentrate the nanoparticles tenfold, as the volume of the lower phase would be 1/10 of the volume of the total sample solution. Note that as the volume ratio becomes more extreme, greater fold improvement is achievable, but the system also requires additional time to separate.

Visualization of ATPS in Paper

After addition of mixed ATPS to the paper membrane, the PEG-deficient phase containing purple dextran coated gold nanoparticles was observed to flow rapidly through the paper. On the other hand, the PEG-rich phase containing blue dye was maintained at the beginning of the paper membrane ( FIG. 34 ). This result was similar to the case where the two phases of ATPS were allowed to safely separate inside the glass well before flow was induced through the paper ( FIG. 80 ). The improved phase separation that occurred within the paper membrane was evident when using 1:1 volume ratio ATPS ( FIG. 34A ), the 1:1 volume ratio ATPS phase separated almost immediately within the paper. As shown in: FIG. 33 , the fold-concentration of purple dextran-coated gold nanoparticles achieved using ATPS is a function of volumetric ratio. Specifically, because the particles are extremely partitioned in the PEG-deficient phase, a 1:1 volume ratio yields twice the concentration of particles when they flow half of their initial volume as the leading front of flow. One possible explanation for the phase separation behavior in paper membranes is that the PEG-rich domains experience more interactions with the paper, allowing them to migrate less. In addition, the PEG-rich domains are also more viscous and thus may experience greater difficulty traveling through tortuous paper networks. In contrast, the PEG-deficient domain interacts less with the paper, is less viscous, allows it to travel quickly through the paper network, and is incorporated at the front of the reading. In the case of a 9:1 volume ratio ATPS, the PEG-deficient domains constitute only 1/10 of the total volume, making it more difficult for them to coalesce and flow ahead of the PEG-rich domains. Specifically, the time required for macroscopic phase separation to occur in the paper was longer than the time it took for the fluid to wick the paper, and no good separation was observed ( FIG. 34B ).

It was also observed that when using both 1:1 and 9:1 volume ratio ATPS, some PEG deficient domains did not do it in the reading front of the flow. This indicated that all, but not all of the PEG-deficient domains were able to escape from the PEG-rich domains, explaining why the in vitro measured equilibrium volume ratios did not match the wicking distances on the paper membrane. To improve LFA, most of the PEG-deficient domains reach the macroscopic PEG-deficient phase and need to be separated from the macroscopic PEG-rich phase before reaching the detection region. Therefore, additional components were needed to further improve the phase separation propensity.

Visualization of ATPS in 3-D Paper Well Devices

Paper wells using 3-D paper structures were designed to further improve the phase separation behavior. Using 3-D paper wells allows the gravity, which normally drives phase separation in vitro, to also assist ATPS phase separation within the paper. The 3-D paper structure also increases the cross-sectional area perpendicular to the flow direction. Thus, more volume can be wicked through the paper simultaneously, allowing more PEG-rich domains to be counter-retained by their interactions with the paper and PEG-deficient domains for easier coalescence. As shown in Fig . 35 , 3-D paper wells contributed to greater phase separation efficiency compared to the previously shown 2-D paper membranes. The PEG-rich domains remained in the top layer of the paper wells, while the PEG-deficient domains containing the dextran coated gold nanoparticles flowed towards the bottom layers of the paper wells. Additionally, the PEG-deficient domains were the first to depart from paper wells and were efficiently separated from the PEG-rich domains. A working buffer was also added to further drive fluid flow and assist flushing of any remaining PEG deficient domains through the paper wells, and it is envisioned that this addition could be automated in the future. Note that the flow is slower when using a 9:1 volume ratio ATPS (solution does not reach the end of the strip after 390 seconds) because there is a large volume of the more viscous PEG rich phase.

Detection of Tf Using a 3-D Paper Well Device

After visualizing improved ATPS phase separation and flow behavior in 3-D paper well devices, we questioned whether LFA could be combined with this technique to improve detection limits. Brilliant Blue FCF dye was deprecated and functionalized anti-Tf DGNPs were used instead of dextran coated gold nanoparticles. DGNPs are cleaved similarly to dextran-coated gold nanoparticles in ATPS, but can capture target biomarkers in samples and function as colorimetric indicators for LFAs.

Detection limits were identified for the LFA-only control without ATPS and 3-D paper well devices. To improve the detection limit of LFA relative to the control in a competition format, the antibody decorated on DGNP needs to bind more Tf. This can be achieved by exposing the same number of DGNPs to a greater number of Tf molecules, and for a fixed concentration of Tf, it is necessary to increase the total volume of the solution. Although DGNPs can be saturated with an increase in total volume, they are diluted with a fold-increase in volume. Since each of the LFA strips can handle only 20 μL of sample due to dilution of DGNP, the nullity test results when not enough DGNP is bound to the control line. However, when using ATPS and 3-D paper well devices, DGNP saturated with Tf molecules is extremely cleaved on PEG depletion and can therefore be concentrated in front of the reading front of the flow to result in validation testing. Therefore, the sample volume was doubled to 40 μL when using a 1:1 volume ratio ATPS, which is expected to induce the same number of DGNPs to enter the detection region by doubling the concentration of DGNPs when compared to the control group. Similarly, the sample volume was increased 10-fold to 200 μL when using a 9:1 volume ratio ATPS expected to enrich DGNP 10-fold. Because only the lower phase of 1:1 and 9:1 volumetric ratio ATPS containing DGNP must pass through the detection region, the volumetric handling capability of the device was fine-tuned by changing the number of layers containing 3-D paper wells. To provide this increase in sample volume, the 3-D paper well used for detection of Tf in a 1:1 volume ratio ATPS solution consisted of 5 layers of paper, whereas for a 9:1 volume ratio ATPS solution, paper 20 increased to the canine layer ( FIGS. 36B&C ), to provide increased volume. Despite the significant increase in the number of layers, the 3-D paper wells for the 9:1 volume ratio ATPS remained relatively small compared to the dime.

When ATPS solutions containing DGNPs are added to the paper wells, the DGNPs rapidly concentrate on the reading front of the solution as they are wicked through the device. DGNP reaches the detection area of the device prior to the remainder of the solution remaining in the paper wells. For a competition assay, the presence of a test line indicates a negative result, whereas the absence of a test line indicates a positive result. When using a negative control containing no model protein Tf, at less than 25 min, both settings using 1:1 and 9:1 volume ratio ATPS rendered visible bands in both test and control lines, respectively, resulting in the absence and validity of Tf. The test is shown ( FIG. 36 ). After confirming that 3-D paper wells can be combined with LFA to accurately assess the absence of Tf in negative control ATPS samples, the Tf concentrations were determined to find limits of detection when using 1:1 and 9:1 volume ratio ATPS solutions. These experiments demonstrated 2-fold and 10-fold improvements in the detection limit of Tf, respectively, compared to conventional LFA ( FIG. 37 ). For a 9:1 volume ratio ATPS experiment using 3-D paper wells, the sample was placed in the device for an additional 10 minutes to allow sufficient time for DGNP to capture the target protein and to phase separate macroscopically prior to addition of the working buffer. was cultured in Because DGNP is more concentrated in mixed ATPS, this incubation period is not required for 1:1 volume ratio ATPS, making DGNP easier to probe the whole solution. These experimental results show that conventional LFA detects Tf at a concentration of 1 ng/μL (a concentration at which no test line appears), whereas this 3-D paper-based diagnostic device has a Tf when 1:1 volume ratio ATPS is used. was detectable at 0.5 ng/μL (2-fold improvement in detection limit). Similarly, the 3-D paper-based diagnostic device was able to detect Tf at 0.1 ng/μL when a 9:1 volume ratio ATPS was used (a 10-fold improvement in detection limit). These results suggest that ATPS solutions with desired volumetric ratios can be combined with well sized 3-D paper wells to significantly and predictably improve biomarker detection using LFA.

Example 4. Detection of M13 bacteriophages in PEG-salt ATPS

To increase the viability of the combination of ATPS and LFA in a point-of-need (PON) setting, the following modifications were made to the system: (1) polyethylene glycol (PEG) and potassium phosphate (salt) ATPS were added to shorter phases. PEG with gold nanoprobe indicators used for LFA by investigating the separation time, (2) testing the kinetic behavior of biomolecular cleavage allowing a significant further reduction in extraction time, and (3) decorating the gold surface with PEG. A custom MATLAB script was created to ensure the compatibility between the salt-rich phases of -salt ATPS and (4) quantify the observed LFA signal intensity. A visual representation summarizing these changes is shown in FIG. 38 .

Culture of bacteriophage M13

Escherichia coli coli ) ( E. coli ) bacteria, strain ER2738 (American Type Culture Collection, ATCC, Manassas, VA) were cultured in 8 mL Luria broth (Sigma Aldrich, St. Louis, MO), 37 °C, incubator shaker. from 6 hours. To culture the model virus bacteriophage M13 (ATCC), a small amount of frozen M13 was added to the bacterial culture. The bacterial cultures were then placed on an incubator shaker at 37 °C for 14 h. The solution was centrifuged at 4 °C and 6000 g for 15 min to isolate M13 in the supernatant from the bacteria in the pellet. The supernatant was extracted from the solution and filtered through a 0.22 μm syringe filter (Millipore, Billerica, Mass.). The dilution of this stock M13 solution was calculated using the concentration of M13 determined by the plaque assay, so the concentration of M13 is reported in plaque forming units/mL (pfu/mL). All reagents and materials, unless otherwise noted, were purchased from Sigma-Aldrich (St. Louis, MO).

Discovery of the volume ratio of PEG-salt systems

For polymer-salt ATPS, polyethylene glycol 8000 (PEG, VWR, Brisbane, CA) and potassium phosphate (5:1 dibasic: monobasic) were combined with Dulbecco's PBS (Invitrogen, Grand Island, NY, pH 7.4, 1.47 mM KH). ₂ PO ₄ , containing 8.1 mM Na ₂ HPO ₄ , 137.92 mM NaCl, 2.67 mM KCl, and 0.49 mM MgCl ₂ ). A final solution was prepared with a final mass of 5 g containing 2% (w/w) Luria broth, consistent with experiments involving M13 samples containing Luria broth. Equilibrium volume ratio (volume of macroscopic upper phase divided by macroscopic lower phase) was determined after a minimum of 12 hours (equilibrium after well) by marking the interface and the outside of the tube at the top of the solution. The tube was then emptied, dried and weighed on a balance. Water was added to the mark and the volumes of both phases were determined using 1 g/mL as the density of water. Different equilibrium volume ratios were found by changing the initial PEG concentration, the initial salt concentration, and keeping the temperature constant at 37 °C. It should be noted that these volume ratios were chosen from the same tie line to ensure similar cleavage of biomolecules, and the specific operating conditions used are listed in Table 2.

Table 2. Tested ratios of PEG-salt ATPS

Cleavage and enrichment of M13

For each concentration experiment, four identical 5 g PEG-salt solutions were prepared. Of these solutions, three were used for the concentration step (n=3), while one was used as a control representing the initial M13 concentration. To ensure that each solution was present in one phase prior to phase separation, all solutions were equilibrated at 4 °C and mixed to homogeneity. M13 was then added to the four solutions to obtain an overall initial concentration of 1x10 ⁸ pfu/mL. To determine how M13 concentrations change with time, 9:1 volume ratio solutions were incubated in a 37 °C water bath for 30 min, 1 h, 4 h, and 24 h. To determine how M13 concentration varies with volume ratio, 9:1, 6:1, 3:1, and 1:1 volume ratio solutions were incubated in a 37 °C water bath for 30 min. In each experiment, the lower phase of 3 in solution was carefully withdrawn using a syringe. The control solution underwent the same incubation steps to mimic the same conditions as the test solution. However, after incubation, the control group was once again equilibrated to 4 °C and mixed to homogeneity before recovering an aliquot to reveal the initial concentration of M13 in ATPS. The M13 concentration of the extracted lower phase and control solutions was determined by plaque assay.

Preparation of gold nanoprobes

Colloidal gold nanoparticles were prepared. The diameter of the gold nanoparticles was then determined using dynamic light scattering (DLS) using a Zetasizer Nano SZ particle analyzer (Malvern Instruments Inc., Westborough, MA). Subsequently, the concentration of gold nanoparticles was determined using Beer's law. The absorbance values were the peak absorbances, and the molar absorption coefficients were taken from the data sheet provided by BB International Life Sciences (see Table 3).

Table 3. Gold Nanoparticle Properties

After quantification of gold nanoparticles, mouse monoclonal antibody (Abcam Inc., Cambridge, MA) to the coat protein pVIII of M13 was administered to nanoparticles at a ratio of 16 μg of anti-M13 per mL of colloidal gold to form gold nanoprobes. added to the solution. The pH of the solution was adjusted to 9.0 using 0.1 M NaOH to promote the coordination bond between the antibody and gold, and the solution was mixed on a shaker for 30 minutes. To provide steric colloidal stabilization when gold nanoprobes are added to the PEG-deficient, salt-rich phase prior to LFA, PEG-2000-SH (Nanocs, Boston, MA) molecules are divided into 5000 PEG-2000-SH molecules versus 1 were bound to the gold nanoprobes in a molar ratio of gold nanoprobes. The solution was mixed for an additional 30 minutes. To prevent non-specific binding, 100 μL of 10% bovine serum albumin (BSA) solution was added to 1 mL of colloidal gold solution to block residual excess surface, and the solution was mixed for 30 minutes. Finally, the solution was centrifuged at 4 °C and 8600 g for 30 min to remove free antibody, PEG and BSA. Decorated gold nanoprobes were resuspended in 150 μL of 0.1M sodium borate buffer at pH 9.0.

Combination of ATPS and LFA

To combine the ATPS pre-concentration step with LFA, varying concentrations of M13 were added to the 9:1 volume ratio PEG-salt solution. After 30 min incubation at 37 °C, the concentrated lower phase was extracted using a syringe. For consistency of immunoassays performed with or without a pre-concentration step, M13 was initially added to the lower phase samples of a 9:1 solution that did not contain M13 to form a control solution. Subsequently, 30 μL of lower phase sample and 10 μL of anti-M13 decorated colloidal gold probe solution were mixed with 30 μL of working buffer. The working buffer solution to aid the flow of the sample through the test strip was adjusted to 0.2% w/v BSA, 0.3% w/v Tween 20, 0.2% w/v sodium azide, 0.1% PEG-8000, and pH 8. in ddH ₂ O and 0.1M Trizma base. To allow the antibody to bind to M13, the resulting solutions were mixed and incubated for 5 min before adding the LFA strips. LFA strips were assembled to run a sandwich assay as schematically illustrated in FIG . 39 . After 10 minutes, the LFA strip was removed and the results were obtained by visually examining the test and control lines on the LFA strip.

Quantitative analysis of LFA

Photos of the LFA strip were taken with a Canon DSLR camera in a controlled lighting environment immediately after the test was completed. To quantify the LFA results, a custom MATLAB script was written. The cropped image was converted to an 8-bit grayscale matrix. Subsequently, the matrix was halved into one production matrix containing the control line and the other containing the test line. For each half matrix, the minimum intensity (darkest dot) was placed along the vector perpendicular to the control or test line. The average position of this minimum was used as the center of a 15 pixel high rectangular area over the length of the control or test line. Subsequently, the average grayscale intensity (I _control ) of all pixels in the control line area was calculated. The same procedure was applied to the other half of the image containing the test line to obtain the average grayscale intensity (I _test ) of the test line area. The average grayscale intensity ( _see I) of a third reference region 15 pixels wide and 50 pixels upstream of the test line was used to normalize the intensity of the test and control lines as follows:

For the control line,

For the test line. Note that high values of intensity correspond to white areas.

biomolecular splitting

Triton X-114 micellar ATPS can be combined with LFA to achieve a 10-fold improvement over the detection limit of LFA virus. The Triton X-114 micellar system takes more than 6 hours to equilibrate the volumes of the two phases. In contrast, the polymer-salt ATPS in this study has a faster phase separation. Also, an initial extraction time of 30 minutes was used for a 9:1 volume ratio.

An aqueous solution composed of PEG and salts, a polymer that participates significantly in hydrogen bonding interactions, will undergo macroscopic phase separation. The PEG-rich, salt-poor phase forms at the top, while the PEG-depleted, salt-rich phase forms at the bottom. Large hydrophilic biomolecules are extremely cleaved on the underlying PEG deficiency. For this example, the model virus bacteriophage M13, which is similar in size and shape to Ebola and Marburg viruses, was investigated. Because M13 has a length of 900 nm and a relatively hydrophilic protein shell, it is expected to be extremely cleavable on the lower PEG-deficient phase due to experiencing fewer repulsive steric exclusion-volume interactions with the PEG polymer on it compared to those on the PEG-rich phase. became In an aqueous two-phase micelle system, M13 is extremely partitioned on a macroscopic micellar deficiency where M13 viral particles also experienced little exclusion-volume interactions.

When the solution is heated and phase separation begins first, PEG rich (M13 deficient) domains and PEG deficient (M13 rich) domains form throughout the entire solution. It is assumed that the viral concentrations in these macroscopic domains reach their equilibrium values very quickly, and the like domains begin to coalesce with each other. As these domains grow larger, their difference in density creates non-uniform gravity and buoyancy forces that cause the PEG-rich domains to rise and the PEG-deficient domains to sink. This leads to the formation of a macroscopic upper, PEG rich (M13 deficient) phase and a macroscopic lower, PEG deficient (M13 rich) phase. The kinetics of this process and the effects of entrained or entrapped domains are further described below.

As the virus is expected to divide extremely in the macroscopic sub-phase, the only concern was in the concentration of biomolecules in the sub-phase upon extraction. The concentration factor is then defined as the concentration of virus, C _virus, sub on the macroscopic subphase divided by the initial total concentration, C _{virus, nascent} .

One potential concern that could negatively affect the concentration coefficient is the presence of entrained PEG-rich (M13 deficient) domains on the macroscopic subphase. Before equilibration, the domains, if not all, had had enough time to travel into their respective macroscopic phases. However, when using an equilibrium volume ratio of 9:1, this is not a problem. Due to the larger volume associated with the PEG-rich domains at the 9:1 volume ratio, it is easier for them to coalesce to form a continuous phase. PEG rich domains have a shorter distance to travel to reach the macroscopic upper phase because the macroscopic lower phase is very small. As a result, there is minimal entrainment of PEG-rich (M13 deficient) domains on the macroscopic subphase, and the concentration of biomolecules is expected to approach its equilibrium value. However, the entrainment of the PEG-deficient (M13 rich) domain is still expected on the macroscopic top. Due to the small volume associated with PEG-deficient domains, it is more difficult to incorporate these domains. They also have to travel an additional distance through the more viscous PEG-rich macroscopic phase to reach the macroscopic lower phase. However, since the main concern is the concentration of biomolecules in the lower phase, and only a small volume is required for LFA, it can be extracted in 30 min and still reach the equilibrium predicted concentration coefficient.

Phase separation of 9:1 volume ratio solutions as a function of time was visually tested. Initially, PEG-rich and PEG-deficient domains are formed throughout the entire solution. The PEG-rich domains coalesce rapidly to form a continuous phase. The solution appears turbid as the entrained PEG-deficient domains cause light to scatter ( FIG. 40A ). Because the PEG-deficient domains coalesce and sink to the bottom due to their large density, a macroscopic bottom, PEG-deficient phase is formed from the bottom stomach. After 30 min, there is a clear macroscopic lower, turbid macroscopic upper, PEG-rich phase that separates from the PEG-deficient phase ( FIG. 40B ). Although the upper phase contains an accompanying PEG-deficient (M13 rich) domain, the main concern is in the turbidity of the lower phase as it contains enriched biomolecules. After a long time, all or most of the PEG-deficient domains migrate into the lower macroscopic phase, and the system is near equilibrium as indicated by the two transparent phases ( FIG. 40C ). Note that the lower phase at 30 min is already transparent, due to the absence of a significant accompanying PEG rich (M13 deficient) domain, suggesting that the measured concentration of virus in it should already reflect its equilibrium value.

To extract the lower phase at 30 min, confirmation that the macroscopic lower, PEG deficient (M13 rich) phase did not contain significant PEG rich (M13 deficient) domains was required. Therefore, the concentration coefficient was determined as a function of time to indicate that the measured concentration of M13 in the lower phase was equal to its equilibrium value even after a short time. Specifically, M13 cleavage experiments were performed using the same temperature and initial PEG and salt concentrations as those used for: FIG. 40 ( ie , conditions to obtain a 9:1 equilibrium volume ratio), lower phase for 30 minutes, 1 Extracted at hours, 4 hours and 24 hours (equilibrium after wells). As shown in Figure 41 , the concentration coefficient remains constant as a function of extraction time from 30 minutes to 24 hours so that the entrainment of the PEG-rich (M13-deficient) domains on the macroscopic lower, PEG-deficient (M13-enriched) phase is practically minimal. prove that Therefore, for follow-up studies, a 30 min extraction time was used as a short time was needed for PON application.

Cleavage of M13 in ATPS

In equation (9), the concentration coefficient can be shown as a function of the volume ratio. Using a molar balance equalizing the sum of the initial moles of virus in a single homogeneous phase and the sum of the moles of virus in the two resulting phases, the following equation was obtained:

where C _{virus, upper} is the concentration of virus in the macroscopic upper phase, and V _upper and V _lower are the volumes of the upper and lower phases _, respectively. Rearranging equation (10) gives the following expression for the concentration factor:

Using a 9:1 volume ratio ATPS, the M13 partition coefficient was determined to be less than 0.0001 (equilibrium concentration in the upper phase divided by the equilibrium concentration in the lower phase). Since this measurement division factor is less than the V _lower /V _upper value 1/9, this equation can be approximated as:

Thus, a linear relationship is predicted for the concentration coefficient as a function of the volume ratio. Because biomolecules are extremely partitioned in the lower phase, the concentration coefficient increases with increasing volume ratio, and the increase in volume ratio corresponds to contraction of the phase in which M13 is extremely partitioned. Specifically, when a 9:1 volume ratio is used, M13 is expected to be enriched tenfold, since the volume of the phase into which M13 is extremely divided is 1/10 of the initial volume. Subsequently, since a short time is required for PON application, it was tested using a 30 min extraction time. M13 cleavage experiments were run at 37 °C using initial concentrations of PEG and salt to obtain 9:1, 6:1, 3:1 and 1:1 equilibrium volume ratios. 42 shows the experimental results of testing the concentration coefficient as a function of volume ratio. To statistically determine whether the experimental data matched the model predictions, coefficients of determination or R-squared values were calculated. The R-squared value was found to be 0.858, suggesting that the experimental data agree reasonably well with the predicted values, further confirming that the simple model is suitable even for a 30 min extraction.

Detection of M13 via LFA

After demonstrating that M13 can be concentrated in 30 min via ATPS, colloidal gold probes and LFA strips were prepared. PEG decorated on the surface of the gold probe provided steric stability and prevented aggregation of the gold probe in the environment of the extracted salt-rich phase. 43 depicts the results of LFA using samples that did not undergo a prior concentration step. The M13 concentration was chosen at regularly spaced points on a logarithmic scale. The presence of the control line or upper band containing the polygonal anti-IgG antibody represents a validated test as it confirms that the sample flowed through the entire strip. The presence of a lower band or test line containing an antibody to the coating protein pVIII of M13 indicates the presence of M13. As shown in the following, negative control: FIG. 43A , contains no M13, and the expected visible test line does not exist. Of the remaining panels, the intensity of the test line is greatest for the highest M13 concentration presented in Fig. 43B , the line intensity decreases with decreasing M13 concentration. The test line is no longer visible at 3.2× ^{10 8} pfu/mL ( FIG. 43E ), showing a detection limit of about 1× ^{10 9} pfu/mL ( FIG. 43D ).

Concentration of M13 prior to LFA

With the detection limit established at 1x10 ⁹ pfu/mL, a possible improvement of the detection limit was investigated by using ATPS. A 9:1 volume ratio solution was used, which was predicted to yield an approximately 10-fold concentration factor based on equation (12). After 30 min incubation at 37 °C, the lower phase was extracted, and samples were prepared for LFA. 44 depicts the results of LFA using a pre-concentration step. Again, the test line is absent for the negative control that does not contain M13 ( FIG. 44A ). Of the remaining panel, the initial M13 concentration is less than 10-fold that shown in FIG. 6 , but the LFA test line intensity is consistent with that of the sample without a prior concentration step. The test line intensity decreases with decreasing M13 concentration until it is no longer visible at 3.2x10 ⁷ pfu/mL ( FIG. 7E ), showing a detection limit of about 1× ^{10 8} pfu/mL ( FIG. 44D ). This indicated a 10-fold improvement in the detection limit when the ATPS concentration step was applied.

To confirm the conclusions from the visual analysis of the LFA strips, the LFA images were converted to grayscale and the test line intensity was tested against the background using this custom MATLAB script. Results are shown in FIG . 45 , with and without the ATPS concentration step. Note that the two curves are separated by a 10-fold difference in the initial M13 concentration for approximately the same test line signal, further confirming the 10-fold concentration improvement of the present invention.

Example 5. Detection of Transferrin in Biological Serum by Interfacial Extraction of PEG-Salt ATPS

Enrichment of biomolecules using a single ATPS step was optimized by driving the target biomolecules towards the interface between the two bulk phases. Because the interfacial region represents a very small volume region that can be formed independent of the volume ratio, this novel approach allows for the enrichment of the target without dependence on extreme volume ratios (volume ratios greater or less than 1) with long phase separation times. makes it possible Instead, the volume ratio that can reach equilibrium the fastest was chosen, which reduces the extraction time in phosphate buffered saline (PBS) to less than 10 min, a significant improvement over previous approaches. This approach shifts towards the maximum fold-concentration achievable in a single ATPS step, since the volume of the interface is smaller than that of the two macroscopic bulk phases, and thus biomolecules can be even more extremely concentrated. Last but not least, increasing the sample volume preferably increases the total number of target biomolecules that can be detected with LFA after being concentrated at the interface. This is also true for using extreme volume ratios to concentrate target molecules in the bulk phase, but increasing the sample volume increases the phase separation time. In the proposed interfacial extraction approach, extreme volumetric ratios are no longer required. 46 graphically compares interfacial extraction with extraction of one of two bulk phases.

To drive target biomolecules towards the interface, we used a capture mechanism involving GNPs. With specific antibodies decorated on the particle surface, GNPs first captured the target protein in the sample. The surface chemistry of GNPs was optimized such that the particles split at the interface upon phase separation. Then, the protein captured by GNP was extracted at the interface. Since the volume of the interface was very small, the protein was highly concentrated and subsequently applied to LFA detection strips. This example provides a summary of the prepared GNPs that were cleavable at the interface of PEG-salt ATPS. Then, the volumetric ratios in which the phases separated the fastest and also allowed the greatest recovery of GNPs were investigated. Subsequently, using the model protein transferrin (Tf), a 100-fold improvement in LFA for Tf combined with an ATPS interfacial extraction step was demonstrated. The study then expanded to approach real-world applications and reoptimized the system for small volumes desirable for fetal bovine serum (FBS) and synthetic urine as well as blood sampling. The data indicate that, even in more complex systems, ATPS interfacial extraction can be performed within 15-25 minutes, leading to a 100-fold improvement in the detection limit of LFA for Tf.

This example provides the development of nanoprobes that can localize at the interface and also function as colorimetric indicators of LFAs. This approach differs from previously studied extraction methods that use functionalized magnetic or gold particles to concentrate and collect target biomolecules, where these approaches required devices that were not suitable for point-of-care devices. This method is an effective and rapid approach to reduce the sensitivity difference between laboratory-based and paper-based immunoassays by improving the detection limit of LFA to 100. Improved LFAs with improved sensitivity are moving closer to providing point-of-care solutions that are not currently available with significant impact in the diagnostic field. On the other hand, it has been demonstrated that this approach can improve LFA detection, and this enrichment procedure can also be applied to other detection methods.

Radiolabeling of anti-Tf antibodies

All reagents and materials, unless otherwise noted, were purchased from Sigma-Aldrich (St. Louis, MO). Iodine-125 ( ¹²⁵ I) was used to radiolabel a goat anti-human Tf polyclonal antibody (catalog # A80-128A, Bethyl Laboratories, Montgomery, TX). Briefly, Na ¹²⁵ I (MP Biomedicals, Irvine, CA) was activated with IODO-BEADS (Pierce Biotechnology, Rockford, IL). Subsequently, activated ¹²⁵ I was reacted with goat anti-Tf antibody for 15 min. The radiolabeled protein was purified and free ¹²⁵ I was removed using a Sephadex G10 size-exclusion column. A phosphotenstic acid assay was used to quantify the radioactivity and concentration of the radiolabeled protein.

Preparation of GNPs

Exposed gold nanoparticles were prepared to result in a clear, cherry-colored solution with a particle size of about 25-30 nm in diameter. To prepare GNP, 320 mg of goat anti-Tf antibody was incubated with 20 mL of colloidal gold solution for 30 min, followed by thiolation- PEG5000 was added. To prevent non-specific binding of other proteins to the surface of colloidal gold, 2 mL of 10% bovine serum albumin (BSA) solution was added to the mixture and mixed for an additional 10 minutes. The resulting solution was gently mixed on a shaker during the incubation period. To remove free (unbound) antibody, PEG and BSA, the mixture was subsequently centrifuged at 4oC and 9,000 g for 30 min. The pellet of GNP was washed twice with 1% BSA solution. Finally, the recovered GNPs were resuspended in 2 mL of 0.1M sodium borate buffer at pH 9.0.

Segmentation of GNP

GNPs decorated with radiolabeled anti-Tf antibody were cleaved in ATPS under different conditions to determine the volume ratio at which the fastest and highest GNP recovery could be obtained. For each split experiment, three identical PEG-salt solutions in Dulbecco's phosphate buffered saline (PBS; Invitrogen, pH 7.4, ionic strength 154 mM) were prepared in a total volume of 5000 μL. PEG-salt ATPS solutions with three different volume ratios (1:1, 6:1 and 1:6) were prepared using specific concentrations of PEG and potassium phosphate. Subsequently, 10 μL of GNPs decorated with radiolabeled anti-Tf antibody were added to each ATPS solution. The solution was equilibrated at 0 ^° C to ensure that the solution was homogeneous. Once equilibrium was achieved at 0 ^o C, the solution was incubated in a 37 ^o C water bath to induce phase separation, and GNPs were found to partition between the two coexisting phases. GNPs from the interface were carefully withdrawn using a pipette, and 30 μL of the interface solution was withdrawn to ensure that most, but not all, of the GNPs were collected from the interface. The two coexisting phases were also withdrawn separately using a pipette. The amount of GNP at the interface and in the two coexisting phases was determined using a Cobra Series Auto-Gamma Counter, since the GNP was bound to the radiolabeled anti-Tf antibody, the amount of radioactivity in each region. was quantified by measuring The quantified amount of GNP in each of the three regions was used to calculate the recovery of GNP at the interface using a mass balance.

Preparation of LFA test strips

An LFA test using a competitive mechanism was conducted in this study ( FIG. 47 ). In a competition assay, the target of interest is immobilized on a nitrocellulose membrane to form a test line. An immobilized secondary antibody to the primary antibody on GNP constitutes the control line. When performing LFA, the sample is first contacted with GNPs, and if target molecules are present in the sample, they bind to their specific antibodies decorated on the GNPs. When a target molecule present in the sample saturates the antibody on the GNP, this GNP can no longer bind to the immobilized target molecule on the test strip. As a result, GNP did not form a visible band in the test line, indicating a positive result. On the other hand, if the sample does not contain the target molecule at a concentration capable of saturating the antibody on the GNP, this antibody on the GNP may bind to the immobilized target molecule on the test strip and form a visible band in the test line. This indicates a negative result. Also, regardless of the presence of the target molecule in the sample, the antibody on the GNP binds to the immobilized secondary antibody on the control line, indicating that sufficient sample has wicked through the test line and reached the control line. The presence of a visible control line indicates a validated test.

Run of LFA with Tf without pre-concentration

Tf stock solutions containing varying concentrations of Tf were prepared in PBS. Subsequently, 20 μL of each Tf stock solution was added to 20 μL of test buffer (0.2% BSA, 0.3% Tween20, 0.2% sodium azide, 0.1% PEG, 0.1 M) used to aid the flow of the sample through the test strip. Trizma buffer, pH 8) and 10 μL of GNP suspension. A total of 5 sample solutions (50 μL each) with varying concentrations of Tf were prepared (0 (negative control), 0.001, 0.01, 0.1 and 1 ng/μL). A test strip was vertically immersed in each sample solution, where the sample pad was in contact with the solution. After 10 minutes, the test strips were withdrawn and images of each strip were immediately taken by: a Canon EOS 1000D camera (Canon U.S.A., Inc., Lake Success, NY).

In experiments run in FBS (HyClone, characterized, pH 7.4), a more concentrated GNP suspension was used so that the volume of GNPs could be reduced to suit small volume experiments. The concentration of Tf in the FBS stock solution was adjusted to achieve the same final Tf concentration used in the PBS experiments by adding 5 μL of the Tf stock solution to 5 μL of the GNP suspension followed by 40 μL of test buffer. Similarly, experiments were performed using synthetic urine.

Combination of ATPS interfacial extraction and LFA for Tf

A volumetric ratio of 1:1 was used for studies performed in PBS based on findings from split GNP experiments. By using an anti-Tf antibody, GNPs were the total Tf-GNP complexes that first captured Tf in the sample and then concentrated at the interface. A protocol similar to that described in the section on cleavage of GNPs was used, except that various concentrations of Tf were also spiked in ATPS solution. Briefly, 10 μL of GNP suspension was obtained in a 1:1 volume ratio and added to 4990 μL of Tf-spiked ATPS solution containing various Tf concentrations (0 (negative control), 0.001, 0.01 and 0.1 ng/μL). The solution was equilibrated at 0 ^° C to ensure that the solution was homogeneous. Once equilibrium was achieved, the solution was placed in a 37 ^° C water bath to induce phase separation. After 10 min, 30 μL of interfacial solution containing concentrated GNP and Tf was withdrawn. This interfacial solution was mixed with 20 μL of test buffer to form 50 μL of sample solution. LFA test strips were vertically immersed in each sample solution, where the sample pad was in contact with the solution. After 10 minutes, the test strips were withdrawn, and images of each strip were immediately taken with a Canon EOS 1000D camera.

For studies performed in FBS and synthetic urine, PEG and potassium phosphate concentrations need to be adjusted first to achieve a 1:1 volume ratio. ATPS in FBS also phase separated more slowly, and instead of the 10 min incubation used in the PBS system, the solution was placed in a 37 ^° C water bath for 25 min. Also, as described above, the volume was reduced. Thus, rather than indicating that the limit of detection is increased using 5000 μL (100-fold greater volume than the 50 μL Tf stock solution used for LFA alone experiments), studies performed in FBS and synthetic urine showed that 1000 μL (used for LFA alone experiments) 100-fold more solution than the 10 μL Tf stock solution) was used to show equivalent improvement. The protocol previously described for PBS was modified for smaller volumes such that 5 μL of a more concentrated gold suspension was added to 995 μL of FBS or a solution of Tf-spiked ATPS in synthetic urine. 20 μL of interfacial region was extracted, then 30 μL of gold buffer was added. Each LFA strip was immersed in suspension for 15 minutes prior to drawing and imaging.

To combine ATPS interfacial extraction with a paper-based LFA detection assay, the GNP developed in this example contained three functions. First, a specific antibody decorated on the surface of the GNP captured the target protein present in the sample. Second, the optimized formation of PEG and protein on the surface of the GNPs allowed the GNPs to split at the interface rather than in the bulk phase. Finally, GNPs functioned directly as colorimetric indicators for LFAs, thus allowing subsequent detection assays to be run immediately without extra washing or other manufacturing steps. A schematic diagram of the GNP is shown in FIG . 48A . GNPs have three main components: PEG polymers, gold nanoparticles, and anti-Tf antibodies. Each component itself drives the nanoparticles into one of two bulk phases. First, the decorated PEG drives the nanoparticles into the upper PEG-rich phase due to the good PEG-PEG interaction between the polymer on the particle surface and the polymer enriched in the upper phase ( FIG. 48B ). Specifically, increasing the molar ratio of PEG:GNP changes the morphology of the bound PEG to more closely resemble the “brush” morphology, thus enlarging the amount of surface area exposed to increase PEG-PEG interactions. On the other hand, the large size of the gold nanoparticles allows the nanoparticles to split on the underlying PEG deficiency, where they experience less repellent exclusion-volume interactions with the PEG polymer. The hydrophilic proteins (anti-Tf Ab and BSA) on the GNPs increase the hydrophobicity of the GNPs, which also allows them to be resolved in the lower PEG-deficient phase, which is more hydrophilic than the upper PEG-rich phase. However, as the underlying PEG-deficient phase is also salt-rich, the nanoparticles aggregate when there is not enough PEG on the surface of the nanoparticles to provide sufficient steric stability ( FIG. 48c ). In combination, the three components of GNP can be varied and delicately balanced to ultimately drive the GNP to the interface in ATPS ( FIG. 48d ).

In competitive LFA, successful detection of a target protein in a sample relies on antibodies on the GNPs encapsulated by the target. If the antibody is not saturated, the GNP can bind the target immobilized on the LFA to form a test line, giving a false negative result. One way to detect a sample with a low concentration of target is to increase the sample volume. It increases the total number of target molecules, which will also potentially lead to saturation of the antibody for a given fixed amount of GNP. However, because only a small volume can flow through the LFA test strip, and because the GNP is diluted in this approach, if not enough GNP flows through the test strip, the control line is not visible, indicating an invalid result. . As the use of ATPS interfacial extraction provides a rapid and effective means to collect GNPs saturated with small volumes of target protein, this approach can result in the detection of low concentrations of target proteins by allowing much larger sample volumes to be analyzed. .

Three volumetric ratios were tested to determine the optimal volumetric ratio to recover most of the GNPs within the shortest incubation period. The results are shown in Table 4.

Table 4. Recovery of GNPs for different volumetric ratios

The 1:1 volume ratio separated the fastest and allowed the greatest recovery of GNPs. When phase separation is induced by increasing the temperature, macroscopic PEG-rich and PEG-deficient domains are formed, and similar domains find and coalesce with each other. When the domains coalesce, they migrate and eventually form a macroscopic PEG-rich, salt-poor phase at the top and a macroscopic PEG-deficient, salt rich phase at the bottom due to the interfacial tension and density difference between the two phases. A 1:1 volume ratio phase separates faster than a 6:1 or 1:6 volume ratio because the domains have an easier time to discover and coalesce with each other when a significant amount of each phase is present. For more non-uniform volumetric ratios, domains in the smaller volumetric phase may accompany the larger continuous phase due to domains experiencing coalescence difficulties. In addition, the 6:1 volume ratio phase is such that the PEG rich phase is the continuous phase for the 6:1 volume ratio, and the PEG-deficient domains experience greater difficulty finding each other and migrating into their respective macroscopic phases in the more viscous PEG-rich continuous phase. Therefore, it separates more slowly than the 1:6 volume ratio.

Table 5: Recovery of GNPs for different volume ratios

Since GNPs do not divide in either domain, they remain between domains when the domains are merged. Eventually, GNPs appear as red thin films at the interface when phase separation is complete. The recovery of GNPs is efficient when using a 1:1 volume ratio, since entrainment is minimized at this volume ratio and thus less GNP is lost at the interface present between entrained domains and continuous phase. This was used for subsequent experiments, as the 1:1 volume ratio phase separated the fastest, yielding the highest GNP recovery.

To demonstrate the improvement of LFA by introducing an ATPS interfacial extraction step, the model protein transferrin (Tf) was used. Tf is a serum protein for iron transport. To establish the detection limits of Tf in LFA, a series of LFA tests using various Tf concentrations were performed without any prior concentration step. If the sample contained enough Tf molecules to saturate the decorated anti-Tf antibody on the GNP, then this anti-Tf antibody did not bind the immobilized Tf on nitrocellulose in the test line, thus forming a visible band in the test line. Did not do it. This resulted in positive results that were observed when samples were tested with a Tf concentration of 1 ng/μL ( FIG. 49 , top panel). On the other hand, if there was insufficient or no Tf in the sample saturating the anti-Tf antibody, this anti-Tf antibody successfully bound to the immobilized Tf on the nitrocellulose membrane, thus forming a visible band in the test line. This is indicative of the negative results observed when samples were tested with Tf concentrations less than 1 ng/μL. As 1 ng/μL is the lowest Tf concentration that gave a truly positive result, it gave a detection limit of about 1 ng/μL for Tf when LFA was performed without a prior concentration step.

To determine that the ATPS interfacial extraction step could improve the detection limit of Tf by 100-fold using LFA, an equal amount of GNP was added to the ATPS solution at a Tf concentration (0.01 ng/μL) that was less than 100-fold the detection limit of LFA. applied to The sample volume was increased 100-fold from 50 μL to 5000 μL to keep the total number of Tf molecules the same. Since only a limited amount of sample (50 μL) can be applied to the LFA test strip, the diluted GNPs in this large sample solution need to be concentrated and applied to the LFA. To recover these GNPs saturated with the target protein, the solution was placed in a 37 ^° C water bath to collect the GNPs within 10 min at the interface. GNPs were then extracted and applied directly to LFA test strips. The results of this study are presented in the following lower panel: FIG. 49 . True positive results were obtained at 0.01 ng/μL indicating a 100-fold improvement in the limit of detection. The test line intensity of a false negative result at 0.001 ng/μL using this approach is weaker than without a pre-enrichment step when the sample is compared to the same Tf concentration, so more Tf is captured to make it difficult for GNPs to bind to the test line. indicates that it has been Test line intensity also increased with decreasing Tf concentration, which was expected when the amount of Tf available to saturate the antibody decreased.

To study the effectiveness of ATPS interfacial concentration, a small volume of ATPS solution prepared with FBS was tested to mimic a small sample blood drawn from a patient. Due to the more complex composition of FBS, the procedure used with ATPS in PBS was reoptimized for the FBS system. The high protein content of FBS altered the volume ratio of ATPS, which required different concentrations of PEG and salt to form a 1:1 volume ratio. Because the experiments performed in FBS also used smaller sample volumes, the volume of GNPs had to be scaled down and a more concentrated GNP stock was prepared. In addition, the incubation time for ATPS was extended from 10 min to 25 min when FBS slowed down the phase separation process. Additionally, due to the complex mixture comprising FBS, the LFA test time was extended from 10 minutes to 15 minutes. Although serum represents a more complex matrix, FIG. 50 shows that LFA combined with ATPS interfacial extraction still yielded a 100-fold improvement in detection limit compared to LFA without prior enrichment. 10 μL of sample for LFA was used and 1000 μL of sample for LFA was combined with ATPS interfacial extraction. A similar optimization process was performed for the synthetic urine system, eventually demonstrating a similar 100-fold improvement in detection limits, as shown in FIG . 51 .

Example 6. Detection of Malaria Biomarkers Using Triton-X-114 ATPS and 3-D LFA

Triton X-114 ATPS was applied to 3-D paper structures to determine if the paper setup could enhance the separation process of this micelle system. The Triton X-114 micellar system phase separates much slower than the PEG-phosphate salt ATPS, resulting in an even greater reduction in phase separation times of at least 8 hours to about 3 minutes, extending this phase separation phenomenon of paper to micellar ATPS. . The paper-based design was then integrated with LFA to form an all-in-one paper-based diagnostic strip that detects disease biomarkers while enriching without user intervention. To demonstrate this, diagnostic strips were prepared from the malaria biomarker Plasmodium falciparum lactate dehydrogenase ( pLDH ) in a solution of phosphate buffered saline (PBS) and undiluted fetal bovine serum (FBS) . ) was used to detect The powerful, one-step automated diagnostic strip is concentrated and detects pLDH in less than 20 minutes, demonstrating a 10-fold improvement over the limit of detection of pLDH when compared to a traditional LFA setup. This platform technology can be used to overcome the above limitations and transform the current status of diagnostics for malaria and other diseases within resource deprivation settings. material and

Preparation of gold nanoprobes (anti-pLDH GNPs)

A solution of gold nanoparticles with an average hydrodynamic diameter of 24 nm was prepared, which appeared as a dark cherry solution. The size of the gold nanoparticles was obtained by dynamic light scattering measurement using a Zetasizer Nano ZS particle analyzer (Malvern Instruments Inc, Westborough, Massachusetts).

After forming nanoparticles, the pH of 1 mL of gold nanoparticle solution was first adjusted to pH 9 using 1.5N NaOH. Subsequently, 16 μL of mouse monoclonal anti-P.falciparum/P.bibox LDH antibody (BBI Solutions, Cardiff, UK) was added to the colloidal gold solution at a concentration of 0.5 mg/mL and placed on a shaker on a shaker. Mix for 30 minutes. To prevent non-specific binding of other proteins to the surface of colloidal gold nanoparticles, 200 μL of a 10% w/v bovine serum albumin (BSA) solution was added to the mixture and mixed on a shaker for 20 minutes. To remove the free unbound antibody, the mixture was centrifuged at 4 °C and 12,000 rpm for 30 min, then the pellet of colloidal gold nanoparticles was resuspended in 200 µL of 1% w/v BSA solution. The centrifugation and resuspension steps were repeated two more times, and after a third centrifugation, the pellet of gold nanoparticles was resuspended in 100 μL of 0.1M sodium borate buffer at pH 9.0. Gold nanoparticles functionalized with anti-pLDH antibodies are hereinafter referred to as anti-pLDH gold nanoprobes (anti-pLDH GNPs). BSA coated gold nanoparticles that were not functionalized with antibody were used for visualization purposes and are hereafter referred to as BSA-GN.

Preparation and visualization of Triton X-114 ATPS

The equilibrium volumetric ratio of Triton X-114 ATPS (the volume of the upper phase divided by the volume of the lower phase) was found by varying the initial w/w concentration of Triton X-114 (Sigma-Aldrich, St. Louis, MO), Dulbecco. Phosphate buffered saline (PBS; Invitrogen, Grand Island, NY, pH 7.4, containing 1.47 mM KH ₂ PO ₄ , 8.1 mM Na ₂ HPO ₄ , 137.92 mM NaCl, 2.67 mM KCl, and 0.49 mM MgCl ₂ ) and FBS ( in solution from Invitrogen, Grand Island, NY). This solution phase separated and reached equilibrium at 25 °C in a temperature controlled water bath. BSA-GN in Triton X-114/PBS solution showed favorable splitting in the upper phase, whereas BSA-GN in Triton X-114/FBS solution split favorably in the lower phase. Consequently, the conditions for a 1:9 volume ratio for Triton X-114/PBS ( ie , the volume of the upper phase divided by the volume of the lower phase) and a 9:1 volume ratio in Triton X-114/FBS were found, and further experiments were carried out. was used for This volume ratio allowed 10-fold concentration of nanoparticles.

To visualize the two phases of micellar ATPS in PBS, 100 μL of BSA-GN and 4 μL of Brilliant Blue FCF dye (The Kroger Co., Cincinnati, OH) were added prior to Triton X-114 for a 1:9 volume ratio in PBS. 2.5 g of solution containing the determined concentration was added. This solution was thoroughly mixed by vortexing and incubated at 25°C. Pictures of the solution were taken every hour until equilibrium was reached at 8 hours. Equilibrium was established when the solution lost its cloudy appearance, all visible domains shifted into their respective phases, and the measured interfacial height remained stable. Cherry BSA-GN and blue dye were colorimetric indicators of micellar-deficient and micellar-rich phases, respectively. All images were captured using a Canon EOS 1000D camera (Canon U.S.A., Inc., Lake Success, NY), in a controlled lighting environment.

To visualize the phase separation of micellar ATPS in PBS in a 3-D paper wick, 200 mg of a mixed homogenous solution containing 2 μL of brilliant blue FCF dye and 10 μL of BSA-GN was vortexed and then placed in a water bath at 25 °C. placed. A 3-D paper wick was formed by laminating three 5 x 15 mm glass fiber paper sheets over one edge of a 5 x 40 mm strip of glass fiber. After the prepared solution was incubated at 25° C. for 5 minutes, a 3-D paper wick was placed vertically in the solution to allow the paper stack to absorb the solution. Images of the paper strip were captured at 0 sec, 30 sec, 60 sec and 180 sec.

Detection of pLDH by LFA alone

LFA test strips using a competitive assay format were assembled. fixed blood. Falciparum L-lactate dehydrogenase (pLDH; MyBioSource, San Diego, CA, USA) constituted a test line and an immobilized goat anti-mouse IgG secondary antibody specific for the primary anti-pLDH antibody ( Bethyl Laboratories, Montgomery, TX) constituted the control line. If sufficient pLDH is present to saturate the GNPs in the sample, the pLDH-GNP complex migrating through the LFA strip will not bind to the immobilized pLDH on the test line, leading to the absence of a visible colored band in the test line. In the absence of pLDH, unoccupied antibody on GNP binds to immobilized pLDH and a colored band is formed in the test line region. Regardless of the presence of pLDH, the antibody on the GNP binds to the secondary antibody immobilized on the control line, forming a visible line, indicating successful sample flow through the strip. Thus, a negative result is identified by two colored bands (one test line and one control line), whereas a positive result is identified by only one colored band in the control line.

To confirm the detection limit of pLDH with LFA alone, anti-pLDH GNP was added to the sample solution and allowed to bind to pLDH present in the sample to form a pLDH-GNP complex. 20 μL of a sample solution consisting of 10 μL of anti-pLDH GNP and 10 μL of known concentration of pLDH in PBS or FBS was added in vitro to 30 μL of working buffer (0.2% BSA, 0.3% polyoxyethylenesorbitan momoraurate (Tween 20)). , 0.1 M Tris buffer, pH 8). An LFA test strip was inserted vertically into the sample solution, which wicked through the strip by capillary action upward towards the absorbent pad. Images of test strips from both PBS and FBS samples were taken after 20 minutes in a controlled light environment.

Detection of pLDH by 3-D paper-based LFA and ATPS

The design of the LFA test strip was slightly modified by the addition of a 3-D paper wick. Specifically, the cellulose sample connected to the nitrocellulose membrane was replaced with a 5 x 20 mm glass fiber pad. On top of this glass fiber pad, three additional strips of 5 x 15 mm glass fiber sheets were laminated to form a total of 4 layers of glass fiber paper. The glass fiber layer was wrapped with: Scotch tape adhesive (3M, St. Paul, MN, USA).

For detection of pLDH in PBS samples, 200 μL of well mixed 1:9 volume ratio ATPS containing 10 μL of anti-pLDH GNP and known concentration of pLDH was added to the test tube. The solution was incubated at 25 °C for 5 min to ensure that the solution became turbid, indicating the initiation of phase separation, and to allow GNPs to capture pLDH in solution. A 3-D wick modified LFA strip was then placed in the mixed ATPS and the fluid was allowed to pass through the 3-D wick towards the absorbent pad. An image of the resulting detection area was captured after 20 minutes.

Detection of pLDH in FBS samples followed a very similar protocol, except that 200 μL of mixed 9:1 volume ratio ATPS was prepared instead. Test conditions, including incubation time and line development time, were consistent with those for PBS samples. An image of the detection area was also captured after 20 min.

Triton X-114 ATPS phase separation in vitro

In a solution of PBS above a certain temperature, Triton X-114 micellar ATPS forms an upper micelle-depleted phase and a lower micelle-rich phase. Molecules present in solution partition between two phases based on their physical and chemical properties, such as hydrophobicity and size. The hydrophilic BSA-GN was extremely partitioned in the micellar-depleted phase driven in large part by repulsive steric excluded volume interactions between nanoparticles and larger, more abundant micelles in the micellar-rich phase. Nanoparticles functionalized with specific antibodies can form complexes with target molecules, which complexes are also highly cleaved on micellar depletion. Non-functionalized BSA-GN, which exhibits a cherry color due to their surface plasmon resonance, was used to visualize the micellar-deficient phase generated after phase separation. In contrast, the Brilliant Blue FCF dye is small and hydrophobic, and thus it is highly resolved in the micellar rich phase. When added to the mixed ATPS solution, a blue colored dye was used to visualize the micellar rich phase resulting after phase separation.

The time required to achieve phase separation varies between two different phase systems. For example, the PEG-phosphate salt ATPS phase separates rapidly for most two-phase systems, and the Triton X-114 micellar ATPS is difficult to achieve macroscopic phase separation equilibrium due to the small density difference and interfacial tension between the two phases. Requires a sufficiently long amount of time. Phase separation times also increase to more extreme volume ratios, eg 1:9 or 9:1, where one phase is proportionally much smaller and the macroscopic domains of that phase experience coalescence difficulties. Images of 1:9 volume ratio ATPS in PBS were taken at 25 °C at specific time points; Complete macroscopic phase separation equilibrium was not achieved until after about 8 hours ( FIG. 53A ). The resulting micellar-deficient phase volume was measured to be 1/10 of the total solution volume, confirming a 1:9 volume ratio.

Using Paper Membrane to Improve Triton X-114 ATPS Phase Separation

It has been hypothesized that multiple layers of paper are essential for improving phase separation in paper. Therefore, a “3-D paper wick” consisting of multiple layers of thin, tightly bonded paper strips was designed to increase the phase separation process of Triton X-114 ATPS. When the 3-D wick was placed upright in the mixed ATPS solution in PBS, the solution flowed vertically up to the strip. Almost immediately after the addition of the strip, the micellar-poor phase containing GNPs flowed rapidly ahead of the slow-moving micellar-rich phase containing the blue colored dye ( FIG. 53B ). Note that separation was already observed within the 3-D wick portion of the strip after about 30 seconds, and a more complete separation appeared 180 seconds (3 minutes) after the fluid had already left the wick.

These experiments show that additional layers of wicks aid in coalescence of macroscopic phase domains and provide an increase in cross-sectional area perpendicular to the flow direction to allow a larger volume to wick through the paper at any given time. Furthermore, the less viscous micellar-deficient domains are expected to migrate and coalesce more rapidly forward in the porous paper, whereas the micellar-rich domains remain inverse and due to their large viscosity and potential beneficial interactions with the paper material. Therefore, it is expected to move more slowly. As a result, the macroscopic separation of the two phases, which has been shown to occur chronologically in vitro, is only observed in paper membranes within minutes. A further advantage of the 3-D paper wick is the handling of larger volumes of ATPS solution. In general, the number of layers in a 3D wick is increased to accommodate large volumes of solution. Optimized factors include strip length, width and number of strips. The design was able to achieve complete phase separation over the length of the wick and minimize the length required for the separated micellar depleted phase running to leave the wick. This paper-based structure could potentially be used to enhance many other two-phase systems that require even longer times to separate.

Enhancement of Triton X-114 ATPS Phase Separation in FBS

In contrast to the PBS solution, instead, Triton X-114 ATPS formed in the FBS sample produced an upper micelle-rich phase and a lower micelle-depleted phase. This may be due to the density difference caused by the additional proteins and salts found in FBS. Instead, because the GNPs are extremely partitioned in the lower phase, it was necessary to adjust the initial Triton X-114 concentration to produce a concentrated lower phase with 1/10 of the total volume, or in other words, a 9:1 volume ratio.

Because the orientation of both phases is reversed in serum, the reversed orientation was studied to determine the effect of phase separation in 3-D paper wicks. In fact, when the 3-D wick was placed in mixed ATPS in FBS, results very similar to those of the PBS experiment were observed. Thus, although the density difference drives the micellar-deficient phase down in FBS in vitro, the micellar-deficient phase still leaves the faster phase flowing to the 3-D wick. Thus, the micellar-deficient phase is the leading front irrespective of the phase orientation determined by the relative phase density in vitro. Thus, the more viscous micellar-rich phase is consistently slow moving and is held inversely by the porous membrane, indicating that the viscosity effect dominates any density effect in the flow behavior of micellar ATPS introduced into the paper.

Improvement of LFA-based detection of pLDH in PBS

Since the micellar-deficient phase containing the enriched GNP flows in front of the reading as the 3-D paper leaves the wick, the wick was attached upstream of the nitrocellulose-based detection region of the LFA paper strip. Doing so allowed a smooth transition from the concentration step to the detection step and eliminated the need for syringe extraction. The design for the integrated paper-based diagnostic strip is shown in FIG . 54A . A 3-D wick (concentrated region) made up of multiple layers of glass fiber paper strips was connected to a nitrocellulose membrane with fixed control and test lines (detection region), which were then used as sinks to drive fluid flow and absorbent. connected to the pad. All components were fixed to the adhesive backing, and the 3-D wick was vertically immersed in the turbid ATPS solution.

In the pLDH detection test, brilliant blue FCF dye was no longer used, and functionalized anti-pLDH GNP was used instead of BSA-GN. Anti-pLDH GNPs exhibit a cleavage behavior very similar to BSA-GNs, but can trap pLDH and function as a colorimetric indicator in LFA.

First, detection limits for the LFA-only control without ATPS and 3-D paper wicks were identified using a total sample volume of 20 μL. Since the LFA-only control was able to successfully detect pLDH at 10 ng μL ⁻¹ but could not successfully detect pLDH at 1 ng μL ⁻¹ , its detection limit was determined to be 10 ng μL ⁻¹ . To show improvement of the detection limit by detecting low concentrations of pLDH, the same number of GNPs used in the LFA-only control were exposed to the same number of pLDH molecules by increasing the total volume of the solution containing the low concentration of pLDH. Addition of ATPS and 3-D wick is expected to concentrate GNPs to a small volume in front of the readings, as volume increase typically dilutes GNPs and leads to a reduction in the amount of GNPs entering the detection region in a given period of time. Effectively, GNPs are exposed to the same amount of pLDH at a low concentration, but then concentrated to a volume similar to that treated with the LFA-only control. When using 1:9 ATPS, which was expected to enrich the GNPs 10-fold, the total sample volume was increased 10-fold (to 200 μL) to ensure that the same amount of GNP entered the detection area when compared to the LFA-only control. .

54B demonstrates the use of a modified LFA device in a negative control test that does not contain pLDH in solution. When the modified LFA strips are immersed in an ATPS solution containing anti-pLDH GNPs in PBS, the GNPs are rapidly concentrated in front of the solution in the 3-D wick segment as evidenced by the dark red color at the 60 sec mark. . The solution flowed readily across the nitrocellulose membrane without the use of additional working buffer. The GNP reached the detection area quickly, while most of the solution remained in the paper wick. A visible band appeared in both control and test line regions within 20 minutes, indicating a valid negative test.

Once a valid negative test was identified, the pLDH concentration was changed to determine the detection limit of the integrated device in a 1:9 volume ratio solution. These experimental results show that conventional LFA detects pLDH at a concentration of 10 ng μL ⁻¹ (which produces a truly positive result), whereas a diagnostic strip incorporating Triton X-114 ATPS and LFA pLDH at 1.0 ng μL ⁻¹ could be detected, which is a 10-fold improvement over the detection limit ( FIG. 55 ).

Improvement of LFA-based detection of pLDH in FBS

These experiments were repeated on FBS samples using modified operating conditions to produce a 9:1 volume ratio. Once again, the 3-D paper-based diagnostic device demonstrated a 10-fold limit of detection improvement by successfully detecting pLDH in FBS at 1.0 ng/μL, whereas the LFA-only control was FBS at 10 ng/μL rather than 1.0 ng/μL. pLDH was successfully detected ( FIG. 56 ). Experiments using integrated paper-based devices in FBS samples demonstrated slower fluid flow through the nitrocellulose-based detection zone. This was likely due to the large initial concentration of Triton X-114 and the presence of other serum components that increased the overall viscosity of the sample. Although control and test line signals were fully developed within 20 min ( FIG. 56 ), only when an extra 10 min was given, the entire micelle-deficient phase flows past the detection area, leading to a reduction in background noise ( FIG. 81 ). As in the case of the above tests in PBS, all tests in FBS did not require any prior dilution in buffer, extraction step or addition of working buffer to aid flow.

By demonstrating that malaria pLDH improves LFA detection in serum, the device could potentially be used to improve the current state of the malaria rapid diagnostic test (RDT). Despite the increasing production and use of malaria RDTs, they in most cases need to be used in conjunction with additional methods such as blood film microscopy to confirm results. Limited sensitivity and variability in ease of use are two factors that prevent LFA from being used as a reliable independent assay in remote malaria-affected settings. Integrated paper-based devices have the potential to address these two issues. The integrated concentration component of the device allows for significant sensitivity improvements. Furthermore, since many of these RDTs require the use of working buffers to aid in dilution and fluid flow in the sample buffer, this diagnostic device demonstrates an improvement in user convenience by eliminating this step.

Example 7. Comparison of LFA Paper

Viscosity can play a role in determining what phase appears in front of the reading.

Interactions between ATPS components and the paper material (hydrophobicity, excluded-volume, electrostatic) may play a role in determining which phases appear in the reading front (on glass fibers, the PPG rich phase is dominated by PPG/salt ATPS, whereas The PEG rich phase is delayed in PEG/salt ATPS; PPG is more hydrophobic than PEG).

Different ATPS on paper were investigated to address how the paper improved the phase separation process. First, it was determined whether phase separation was observed in the paper. If so, the image that appeared in front of the reading was observed. Since the reading front corresponds to the phase reaching the test line in LFA, it is desirable to have a phase containing the target molecule and nanoprobes enriched.

Table 6. Combinations of ATPS systems and LFA paper types

In paper, phase separation depended on different ATPSs ( eg PEG/salt vs. PPG/salt).

Different paper materials changed the phase separation behavior ( eg glass fiber vs. PEG/salt in cellulose).

In some cases, the more viscous phase was delayed ( e.g. , for glass fiber materials, the more viscous PEG-rich phase was delayed for the PEG/salt system, whereas the more viscous dextran-rich phase was delayed for the PEG/dextran system). delayed for). However, this was not always the case.

In this example, changing the concentration of polymers in ATPS did not appear to alter the order in which the phases dominate.

In this example, gravity did not appear to alter the order of the flowing phases on the paper ( e.g. , the salt rich phase was the lower phase in PEG/salt ATPS when it phase separated in the tube, whereas the salt rich phase was or it will be the leading front on the glass fiber, regardless of whether it is oriented horizontally).

To investigate how the paper promotes and enhances the phase separation of ATPS, the same type of ATPS (PEG/salt) was applied to different types of paper. Different results were observed.

observe

It was observed that applying sufficiently mixed ATPS to various types of paper did not yield similar results. Instead, the rate of phase separation varied significantly as well as the degree of phase separation. More surprisingly, certain paper types were able to reverse the order in which the phases appeared in the reading fluid. See also Fig. 58 and Table 7.

Table 7. Combinations of ATPS & LFA

Example 8. Some Methods for Making LFA Paper

A known concentration of solute in solvent was added to the paper by micropipette at a value of 40 uL (solvent) per cm ² (glass fiber paper with a height of approximately 1-2 mm). Droplets were added at points evenly distributed over the paper. After addition, the paper was gently rolled (using a pipette tip) into a cylindrical object to help distribute the fluid evenly within the paper.

The solution is pipetted onto a non-absorbent surface ( eg poly-propylene) that forms droplets. The paper segment was contacted with the droplet at a point, edge or surface of the paper segment. This method is useful for making unique specific designs of dewatering. For an example of such a design, see FIGS . 59A-D.

Example 9. Applying a blood sample with a PEG-rich phase to LFA paper

A first experiment was performed to find a paper that could hold a blood sample back without clogging the paper to allow the two phases of ATPS to flow forward. The following experiments were successfully performed, which made it possible to integrate an ATPS enrichment system using MF1 paper used to filter red blood cells.

A 1:1 PEG-salt solution was prepared. The upper PEG rich phase was removed. The PEG rich phase was applied onto filter paper strips along with whole blood samples.

The PEG rich phase was still able to migrate through the filter paper. Blood does not clot the filter paper. MF1 was uniquely suited to ATPS. See also Fig. 71.

Other paper types that cannot filter red blood cells or pass the ATPS phase were investigated. In LF1 and S17, blood was not filtered. In MF1, blood was filtered, but the flow was slow. See also Fig. 72 .

A second experiment was performed to obtain a species composition and type that allowed phase separation and enrichment of plasma in the species' stomach, while retaining blood cells again. Below are two demonstrations of the ability of the 3D paper well design to filter red blood cells while passing through the ATPS phase.

First, blood samples with PEG-salt ATPS were applied to paper with 3-D paper wells. A 9:1 PEG-salt ATPS was prepared and a small volume of whole blood sample was introduced into the PEG-salt solution. Flow strips were prepared with 3-D paper wells with different amounts of paper components at the time of initiation. A blue dye is used to visualize the PEG-rich phase and ensure that the liquid has wicked the strip. The PEG-salt solution, now containing the whole blood sample, was applied to the 3-D wells. Separation and concentration were observed.

The blue liquid front was observed to flow forward. Blood was left behind in the 3-D wells. See also Fig. 73 .

Second, blood samples with PEG-salt ATPS were applied to filter paper with 3-D paper wells. A 9:1 PEG-salt ATPS was prepared and a small volume of whole blood sample was introduced into the PEG-salt solution. Blue dye was used to visualize the PEG rich phase. Gold nanoparticles were used to visualize the salt rich phase. The PEG-salt solution, now containing the whole blood sample, was applied to the 3-D wells and the separation and concentration were observed.

Conditions in which PBS was added instead of gold nanoparticles were also performed.

A blue liquid front was observed wicking through the well. In addition to red blood cell retention, there was a more pronounced blue phase in the ATPS strips compared to the control strips suggesting phase separation. See also Fig. 74 .

A configuration was preferred such that the blood cells were filtered prior to contacting the sample with the ATPS component and concentrating the target in the reading fluid. This alternative configuration would be useful in scenarios where red blood cells affect the phase separation behavior. One experimental setup that has been successful for this purpose is to stratify filtered and concentrated paper types within 3-D paper wells. It was observed that blood is retained again by transferring the PEG phase first. See also Fig. 75 .

Example 10. Chlamydia ATPS-LFA detection in trachomatis

ATPS and LFA were introduced into a single paper-based diagnostic device used to detect Chlamydia trachomatis in a solution of virus transport medium used for storage of the obtained swab samples.

Hang-Mr. Preparation of Trachomatis DGNP

The pH of the 1 mL dextran-coated gold nanoparticles (DGNP) solution was first adjusted to pH 9 using 1.5N NaOH. Then 16 μg of mouse monoclonal Chlamydia trachomatis antibody was added to the colloidal gold solution and mixed on a shaker for 30 minutes. To prevent non-specific binding of other proteins to the surface of colloidal gold nanoparticles, 200 μL of 10% w/v bovine serum albumin (BSA) solution was added to the mixture and mixed on a shaker for 20 minutes. To remove free unbound antibody, the mixture was centrifuged at 4 °C and 9,000 rpm for 30 min, then the pellet of DGNP was resuspended in 200 µL of 1% w/v BSA solution. The centrifugation and resuspension steps were repeated two more times, and after the third centrifugation, the pellet of DGNP was resuspended in 100 μL of 0.1M sodium borate buffer at pH 9.0.

Detection using LFA

LFA test strips using the sandwich assay format were assembled in a manner similar to our previous work. In this format, an immobilized Chlamydia trachomatis antibody constituted a test line and an immobilized second antibody specific for a first Chlamydia trachomatis antibody constituted a control line.

Seeds using LFA. To confirm the detection limit of trachomatis , DGNP was added to the sample solution and C. present in the sample . Trachomatis was combined. The sample solution consisted of DGNP and a known concentration of seed in virus transport medium (BD, Franklin Lakes, NJ). Trachomatis was mixed in vitro. An LFA test strip was inserted vertically into the sample solution, which wicked through the strip by capillary action upward towards the absorbent pad. Images of test strips from both PBS and FBS samples were taken after 10 minutes in a controlled light environment.

Seed. Trachomatis antigen was detected in solution of virus transport medium. See also FIG . 76 . The conjugate used was C. Dextran-coated gold nanoprobes (DGNPs) with antibodies specific for lipopolysaccharide (LPS) on trachomatis antigen. Using the conventional LFA sandwich format, the detection limit of the setup was determined to be approximately 10 μg/mL. Subsequent steps may be to enrich the biomarker using suitable ATPS, apply the enriched sample to LFA, and establish the detection limits of the integrated system.

Seed in PBS. The detection limit of the trachomatis antigen was also determined to be 10 μg/mL using the same LFA settings.

Detection of antigen using DGNP complexed with Abcam's anti-MOMP antibody was also tested. They did not appear to successfully detect antibodies in PBS or VTM solutions and were therefore ignored.

Example 11. ATPS-LFA detection of Streptococcus mutans

ATPS and LFA are the dominant bacteria that can induce caries (cavities), Streptococcus introduced into a single paper-based diagnostic device used to detect mutans .

anti-S. Preparation of mutans DGNP

The pH of the 1 mL dextran-coated gold nanoparticles (DGNP) solution was first adjusted to pH 9 using 1.5N NaOH. Then , 16 μg of mouse monoclonal S. Mutans antibody was added to the gold solution and mixed on a shaker for 30 minutes. To prevent non-specific binding of other proteins to the surface of colloidal gold nanoparticles, 200 μL of 10% w/v bovine serum albumin (BSA) solution was added to the mixture and mixed on a shaker for 20 minutes. To remove free unbound antibody, the mixture was centrifuged at 4 °C and 9,000 rpm for 30 min, then the pellet of DGNP was resuspended in 200 µL of 1% w/v BSA solution. The centrifugation and resuspension steps were repeated two more times, and after the third centrifugation, the pellet of DGNP was resuspended in 100 μL of 0.1M sodium borate buffer at pH 9.0.

Detection using LFA

LFA test strips using the sandwich assay format were assembled in a manner similar to our previous work. In this format, the fixed S. The mutans antibody constituted the test line, and the immobilized secondary antibody specific for the primary antibody constituted the control line.

S. using LFA . To confirm the detection limit of mutans , DGNP was added to the sample solution , and S. to bind to mutans . The sample solution consists of DGNP and contains known concentrations of S. mutans were mixed in vitro. An LFA test strip was inserted vertically into the sample solution, which wicked through the strip by capillary action upward towards the absorbent pad. Images of the test strips were taken after 10 minutes in a controlled lighting environment.

Detection using LFA with ATPS

A 1:9 Triton X-114 micellar ATPS sample solution was prepared, which contained a known concentration of S. It is made up of mutans . The ATPS sample solution was incubated at 25 °C for 8 hours to allow phase separation to occur. Concentrated S. Extract the upper micelle-deficient phase containing mutans and anti- S . Incubated with mutans DGNP. LFA test strips were inserted vertically into the resulting mixture, and images of the test strips were taken after 10 minutes in a controlled lighting environment.

A 9:1 PEG/potassium phosphate ATPS sample solution was prepared, which contained a known concentration of S. It is composed of mutans . The ATPS sample solution was incubated at 25 °C for 30 min to allow phase separation to occur. Concentrated S. The lower PEG-deficient phase containing mutans was extracted, and the anti- S . Incubated with mutans DGNP. LFA test strips were inserted vertically into the resulting mixture, and images of the test strips were taken after 10 minutes in a controlled lighting environment.

s . Mutans were successfully detected using LFA (see FIGS. 77 & 78 ). Using the conventional LFA sandwich format, the detection limit of the setup was determined to be approximately 1x10 ⁷ cells/mL. A 10-fold improvement in detection limits of 1x10 ⁷ to 1x10 ⁶ cells/mL was also demonstrated.

The next step is to introduce ATPS and LFA to the paper in 3D well or 3D wick format, and achieve simultaneous seamless concentration and detection.

Example 12. ATPS-LFA detection of troponin

ATPS and LFA were introduced into a single paper-based diagnostic device used to detect the biomarker troponin for myocardial infarction.

Preparation of anti-troponin DGNP

The same procedure as in Example 11 is used except for the anti-troponin antibody.

Detection using LFA

LFA test strips using the competitive assay format were assembled in the same manner as our previous studies. In this format, immobilized troponin constituted the test line and immobilized secondary antibody specific for the primary antibody on DGNP constituted the control line.

To confirm the detection limit of troponin using LFA, DGNP was added to the sample solution and allowed to bind to troponin present in the sample. Sample solutions containing DGNP and known concentrations of troponin were mixed in vitro. An LFA test strip was inserted vertically into the sample solution, which wicked through the strip by capillary action upward towards the absorbent pad. Images of test strips from PBS samples were taken after 10 minutes in a controlled light environment.

Detection using LFA with ATPS

A 9:1 PEG/potassium phosphate ATPS sample solution was prepared, which consisted of known concentrations of troponin . The ATPS sample solution was incubated at 25 °C for 30 min to allow phase separation to occur. The lower PEG-deficient phase containing concentrated troponin was extracted and incubated with anti-troponin DGNP. LFA test strips were inserted vertically into the resulting mixture, and images of the test strips were taken after 10 minutes in a controlled lighting environment.

troponin was successfully detected using LFA (see FIG. 79 ). Using the conventional LFA competition format, the detection limit of the setup was determined to be approximately 1 ng/μL. A 10-fold improvement for detection limits of 1 to 0.1 ng/μL was successfully demonstrated.

Example 13. how to dehydrate

liquid application

The method involved applying a liquid solution, which contains the desired component to be dehydrated as a solute to the paper prior to dewatering.

A known concentration of solute in solvent was added to the paper by micropipette at a value of 40 uL (solvent) per cm ² (glass fiber paper with a height of approximately 1-2 mm). Droplets were added at points evenly distributed over the paper. After addition, the paper is gently rolled into a cylindrical object (we use a pipette tip) to help distribute the liquid evenly within the paper. The solution was pipetted onto a non-absorbent surface ( eg poly-propylene) that formed droplets. The paper segment was contacted with the droplet at a point, edge, or surface of the paper segment. This method was desirable when creating a unique specific design of dewatering. An example of such a design is presented in FIGS. 59A-D .

Failed Method: Immersion of the entire paper segment in the solvent supersaturates the paper, providing reduced fastness of the experiment, which is likely due to non-uniform dehydration. Additionally, adding more than 60 μL (solvent) per cm ² (glass fiber paper with a height of approximately 1-2 mm) also supersaturates the paper segments.

freeze drying

This method was preferred when the desired result was to redissolve the components. Freeze dryers have recently been found to be important to thaw prior to dehydration. Failure to do so increased the risk of non-uniform dehydration of components within the paper ( eg , dextran-coated gold nanoprobes (DGNPs) would have a greater distribution near the edges of the paper).

flash Freezing and lyophilization

Paper segments were flash frozen using liquid nitrogen by either immersing the paper in liquid nitrogen or pouring liquid nitrogen into the paper. The frozen paper segments were then quickly placed in a lyophilizer where the frozen liquid was sublimed. The reason for trying this method was to try to reduce the movement of solutes during the dehydration process to ensure more control over the dehydration process (as opposed to the solutes that migrate during evaporation of the liquid state solution). However, this process resulted in unexpected flow patterns and phase separation behavior. Below is the time-lapse image data of the unexpected data (see Fig. 60) .

vacuum chamber

Similar to 'lyophilization' described above, except that the paper segments are placed in a vacuum chamber instead of a lyophilizer. The lower pressure in the vacuum chamber demonstrated less consistent experimental results, likely due to the inhomogeneous solute distribution during dehydration.

baking

The paper segments were placed in an oven chamber at a temperature above 25°C (typically 60°C). This method may be desirable when the desired goal is to re-dehydrate the solute while preventing its mobility. One possible explanation for this observed effect is that higher temperatures during the baking process may induce covalent bonds or strong interactions between the solute and paper.

An unsuccessful attempt in a dehydration method that results in non-uniform phase separation as a result of non-uniform dehydration see FIG . 61 .

Claims (26)

A device for detecting and/or quantifying a target analyte in a sample, comprising:
a. lateral flow assays (LFA) involving porous matrices; and
b. An aqueous two-phase system (ATPS) disposed within said porous matrix, comprising:
The porous matrix allows ATPS or a component thereof to flow through the porous matrix when ATPS or a component thereof is present in the fluid phase and facilitates the separation of the mixed phase of ATPS into a first phase solution and a second phase solution within the porous matrix. An aqueous two-phase system (ATPS) configured to provide and having sufficient porosity therefor. The method of claim 1 , wherein the target analyte is contacted with a mixed phase solution, and the target analyte is partitioned into a first phase solution or a second phase solution within the porous matrix, or
wherein the target analyte is contacted with the mixed phase solution and the target analyte is partitioned at the interface of the first phase solution and the second phase solution within the porous matrix. The method of claim 1 , wherein the first phase solution comprises a micellar solution and the second phase solution comprises a polymer or a salt, or
wherein the first phase solution comprises a first polymer and the second phase solution comprises a second polymer. 4. The device of claim 3, wherein the first/second polymer is selected from polyethylene glycol, polypropylene glycol, dextran, and combinations thereof. Device according to claim 1 , wherein the first phase solution comprises polyethylene glycol or polypropylene glycol and the second phase solution comprises potassium phosphate. 5. The device of any one of claims 1 to 4, wherein the target analyte is selected from the group consisting of proteins, antigens, biomolecules, small organic molecules, sugar moieties, lipids, nucleic acids, sterols, and combinations thereof. 5. The device of any one of claims 1 to 4, wherein the device further comprises a probe, wherein the probe interacts with the target analyte. 8. The device of claim 7, wherein the probe comprises a material selected from the group consisting of synthetic polymers, metals, minerals, glass, quartz, ceramics, biological polymers, plastics, and combinations thereof. 9. The method of claim 8, wherein the probe comprises a biological polymer selected from the group consisting of dextran, polypropylene glycol, polyethylene glycol, and combinations thereof, or
the probe comprises a metal selected from the group consisting of gold, silver, platinum, and combinations thereof, or
wherein the probe comprises nanoparticles. The device of claim 8 , wherein the probe comprises a coating, or the coating comprises polypropylene glycol or polyethylene glycol. The device of claim 10 , wherein the coating has affinity for the first phase solution or the second phase solution, and wherein the probe further comprises a binding moiety that binds to the target analyte. The device of claim 11 , wherein the binding moiety is selected from the group consisting of antibodies, antibody fragments, lectins, proteins, glycoproteins, nucleic acids, small molecules, polymers, lipids, and combinations thereof. The device of claim 8 , wherein the probe comprises a detectable label, wherein the detectable label is selected from the group consisting of a colorimetric label, a fluorescent label, an enzymatic label, a colorigenic label, a radioactive label, and combinations thereof. . 5. The device of any one of claims 1 to 4, wherein the LFA comprises a target analyte capture moiety, wherein the target analyte capture moiety interacts with the target analyte. 5. The device of any one of claims 1 to 4, wherein the LFA comprises a probe capture moiety, wherein the probe capture moiety interacts with the probe or component thereof. Device according to any one of the preceding claims, wherein the components of the first phase solution and/or the components of the second phase solution are dehydrated on and/or in the porous matrix. A method of detecting and/or quantifying a target analyte in a sample comprising the steps of:
a. applying the sample to a device for detecting and/or quantifying a target analyte in the sample, the device comprising:
i. lateral flow assays (LFA) involving porous matrices; and
ii. An aqueous two-phase system (ATPS) disposed within said porous matrix, comprising:
The porous matrix allows ATPS or a component thereof to flow through the porous matrix when ATPS or a component thereof is present in the fluid phase and facilitates the separation of the mixed phase of ATPS into a first phase solution and a second phase solution within the porous matrix. an aqueous two-phase system (ATPS) configured to provide and having sufficient porosity therefor; and
b. detecting the presence or absence of the target analyte and/or quantifying the target analyte on the LFA. 18. The method of claim 17,
the target analyte is contacted with the mixed phase solution and the target analyte is partitioned into a first phase solution or a second phase solution, or
wherein the target analyte is contacted with the mixed phase solution and the target analyte is partitioned at an interface of the first phase solution and the second phase solution. 18. The method of claim 17,
the first phase solution comprises a micellar solution and the second phase solution comprises a polymer or salt, or
The method of claim 1, wherein the first phase solution comprises a first polymer and the second phase solution comprises a second polymer. The method of claim 19 , wherein the first/second polymer is selected from polyethylene glycol, polypropylene glycol, dextran, and combinations thereof. 21. The method according to any one of claims 17 to 20, wherein the first phase solution comprises polyethylene glycol or polypropylene glycol and the second phase solution comprises potassium phosphate. 21. The method of any one of claims 17-20, wherein the target analyte is selected from the group consisting of proteins, antigens, biomolecules, small organic molecules, sugar moieties, lipids, nucleic acids, sterols, and combinations thereof.
delete delete delete delete

Images